ES2547305T3 - Polynucleotides, polypeptides encoded by them, and methods of using them to increase tolerance to abiotic stress and / or biomass and / or yield in plants that express them - Google Patents

Abstract

A method to increase biomass, growth rate, seed yield and / or tolerance to abiotic stress of a plant compared to biomass, growth rate, seed yield and / or tolerance to abiotic stress of a non-transgenic plant, in which abiotic stress is selected from the group consisting of osmotic stress, saline stress and nitrogen deficit, the method comprising overexpression in the plant of an exogenous polynucleotide encoding the polypeptide SEQ ID NO. : 201 or an ortholog thereof comprising an amino acid sequence having a homology of at least 80% with the amino acid sequence set forth in SEQ ID NO: 201, wherein said polypeptide is capable of increasing biomass, the rate of growth, seed yield and / or tolerance to abiotic stress of a plant, in which abiotic stress is selected from the group consisting of osmotic stress or, saline stress and nitrogen deficit, wherein said exogenous polynucleotide is comprised in a nucleotide acid construct that additionally comprises a promoter to direct transcription of said exogenous polynucleotide in a cell of a plant, said promoter being a constitutive promoter, a specific tissue promoter, or a promoter inducible by abiotic stress, thereby increasing biomass, growth rate, seed yield and / or tolerance to the abiotic stress of the plant compared to biomass, the rate of growth, seed yield and / or abiotic stress tolerance of the non-transgenic plant.

Description

Polynucleotides, polypeptides encoded by them, and methods of using them to increase tolerance to abiotic stress and / or biomass and / or yield in plants that express them

Field and background of the invention

The present invention, in some of its embodiments, relates to polypeptides and isolated polynucleotides and more particularly, to methods of using them to increase a plant's tolerance to abiotic stress, growth, biomass, vigor and / or yield of a plant. plant.

Abiotic stress conditions (EAB; also called "environmental stress"), such as salinity, drought, flood, suboptimal temperature and toxic chemical pollution, cause substantial damage to agricultural plants. Most plants have developed strategies to protect them against these conditions. However, if the severity and duration of the stress conditions are too great, the effects on development, growth and performance are very pronounced. In addition, most crop plants are very susceptible to EAB and therefore require optimal growth conditions for commercial crop yields. Continuous exposure to stress causes major alterations in plant metabolism eventually leading to cell death and as a result of loss of performance. Therefore, despite carrying out extensive research and taking strong measures regarding crop protection, annual losses of billions of dollars continue annually due to abiotic stress conditions.

Drought is a gradual phenomenon, which implies periods of abnormally dry weather that persists for a long enough time to produce serious hydrological imbalances, such as crop damage and shortage of hydrological reserves. In severe cases, drought can last for many years and produce devastating effects on agriculture and hydrological reserves. With the increasing population increase and the chronic shortage of fresh water available, drought is not only the main problem related to climate in agriculture, but also stands out as one of the main natural disasters of all time, causing no only economic damages (for example, losses due to drought in the United States in 1988 exceeded 40 billion dollars), but also loss of human lives, as occurred between 1984 and 1985 with the drought in the Horn of Africa, which led to a famine that killed 750,000 people. In addition, drought is associated with increased susceptibility to various diseases.

For most crop plants, the world's agricultural regions are too arid. In addition, the excessive use of available water produces an increased loss of agriculturally usable land (desertification), and an increase in the accumulation of salts in soils, added to the loss of water available in them.

Salinity, high levels of salt, affects one of five hectares of irrigated land. It is expected that this condition will only worsen, further reducing the availability of arable land and crop production, since none of the five best food crops, i.e. wheat, corn, rice, potatoes and soybeans, can tolerate excess salt . The harmful effects of salt on plants produce water deficit that leads to osmotic stress (similar to drought stress) and the effect of excess sodium ions on critical biochemical processes. As with freezing and drought, high salt content causes water deficit; and the presence of high salt content makes it difficult for the roots of plants to extract water from their surroundings. Soil salinity is therefore one of the most important variables that determine whether a plant can thrive. In many parts of the world, large areas of land are not cultivable due to a naturally high salinity in the soil. Therefore, the salinization of soils used for agricultural production is a significant problem and is increasing in regions that depend heavily on agriculture and is getting worse due to overuse, fertilization and water scarcity, typically caused by climate change and the demands of the growing population. Salt tolerance is of particular importance at the beginning of the life cycle of plants, since evaporation of the soil surface causes an upward movement of water and salt accumulates in the upper soil layer where the seeds are arranged . On the other hand, germination normally occurs at a saline concentration that is higher than the average saline level in the entire soil profile.

The germination of many crops is temperature sensitive. A gene that could enhance germination in warm conditions could be useful for crops that are planted in a late season or in hot climates. In addition, seedlings and mature plants that are exposed to excess heat can suffer thermal shock, which can originate in various organs, including the leaves and particularly the fruits, when perspiration is insufficient to overcome heat stress. Heat also damages cell structures, including organelles and cytoskeleton and alters membrane function. Thermal shock can cause a decrease in the overall synthesis of proteins, accompanied by the expression of heat shock proteins, for example, chaperones, which are involved in the refolding of heat-denatured proteins.

Heat stress is often accompanied by conditions of low water availability. The heat itself is seen as an interrelated stress and added to the harmful effects caused by deficit conditions

water Evaporative water demand shows almost exponential increases with increases in daytime temperatures and can produce high rates of perspiration and low water potentials for plants. High temperature pollen damage almost always occurs along with drought stress and rarely occurs in well hydrated conditions. Combined stress can alter the metabolism of plants in new ways; therefore the understanding of the interaction between different stresses can be important for the development of strategies to enhance stress tolerance through genetic manipulation.

Excessive cooling conditions, for example, low temperatures, although above freezing temperatures, affect crops of tropical origin, such as soybeans, rice, corn and cotton. Typical damage by cooling includes wilting, necrosis, chlorosis or ion filtration of cell membranes. The underlying cooling sensitivity mechanisms are not yet fully understood, but they probably involve the level of membrane saturation and other physiological deficiencies. For example, photoinhibition of photosynthesis (interruption of photosynthesis due to high light intensities) often occurs in clear atmospheric conditions after cold nights at the end of summer / fall. In addition, cooling can lead to production losses and lower product quality through delayed maturation of corn.

Water deficit is a common component of many plant stresses. The water deficit occurs in the cells of the plants when the entire rate of transpiration of the plant exceeds water collection. In addition to drought, other stresses, such as salinity and low temperature, cause cell dehydration.

Signal transduction by saline stress and drought consists of signaling routes of ionic and osmotic homeostasis. The ionic aspect of saline stress is signaled by the SOS pathway in which a calcium-sensitive SOS3-SOS2 protein kinase complex controls the expression and activity of ion transporters such as SOS1. The osmotic component of saline stress involves complex reactions in plants that overlap with responses to drought and / or cold stress.

Common aspects of response to stress caused by drought, cold and salt [Revised in Xiong and Zhu (2002) Plant Cell Environ. 25: 131-139] include: (a) transient changes in cytoplasmic calcium levels early in the signaling event [Knight, (2000) Int. Rev. Cytol. 195: 269-324; Sanders et al. (1999) Plant Cell

11: 691-706]; (b) signal transduction by calcium dependent protein kinases (CDPK) and / or activated by mitogen and protein phosphatases [Merlot et al. (2001) Plant J. 25: 295-303; Tahtiharju and Palva (2001) Plant J. 26: 461-470]; (c) increases in abscisic acid levels in response to stress triggering a subset of responses; (d) inositol phosphates as signaling molecules (at least for a subset of the stress-sensitive transcripts changes [Xiong et al. (2001) Genes Dev. 15: 1971-1984]; (e) activation of phospholipases that in turn generate a diverse series of secondary messenger molecules, some of which could regulate the activity of stress-sensitive kinases (for example, phospholipase D; Frank et al. (2000) Plant Cell 12: 111-124];

(f) induction of type genes (LEA, Late Embryogenesis Abundant) abundant in late embryogenesis, including COR / RD genes sensitive to CRT / DRE; (g) increased levels of compatible antioxidants and osmolytes, such as proline and soluble sugars [Hasegawa et al. (2000) Annu. Rev. Plant Mol. Plant Physiol 51: 463-499)]; and (h) accumulation of reactive oxygen species such as superoxide, hydrogen peroxide and hydroxyl radicals.

The biosynthesis of abscisic acid is regulated by osmotic stress in multiple stages. Osmotic stress, both dependent and independent of ABA, signals by first modifying the constitutively expressed transcription factors, which leads to the expression of early response transcriptional activators, which then activate effector genes for stress tolerance downstream.

Various genes that increase tolerance to cold or salt stress can also improve drought stress protection, these include, for example, the transcription factor AtCBF / DREB1, OsCDPK7 (Saijo et al. 2000, Plant J. 23: 319-327) or AVP1 (a vacuolar pyrophosphatase proton pump, Gaxiola et al. 2001, Proc. Natl. Acad. Sci. USA 98: 11444-11449).

The development of stress-tolerant plants is a strategy that has the possibility of solving or mediating at least some of these problems. However, traditional plant reproduction strategies used to develop new plant lines that exhibit EAB tolerance are relatively ineffective since they are tedious, slow and with unpredictable results. In addition, limited germplasm resources for stress tolerance and incompatibility at crossings between distantly related plant species represent significant problems encountered in conventional reproduction. Additionally, the cellular processes that lead to EAB tolerance are complex in nature and involve multiple mechanisms of cellular adaptation and numerous metabolic pathways.

Attempts to modify genetic engineering have been described in the art, intended to confer tolerance to abiotic stress to transgenic crops. All studies conducted by Apse and Blumwald (Curr Opin Biotechnol.

13: 146-150, 2002), Quesada et al. (Plant Physiol. 130: 951-963, 2002), Holmström et al. (Nature 379: 683-684, 1996), Xu et al. (Plant Physiol 110: 249-257, 1996), Pilon-Smits and Ebskamp (Plant Physiol 107: 125-130, 1995) and Tarczynski et al. (Science 259: 508-510, 1993) have tried to generate stress-tolerant plants.

In addition, various United States patents and patent applications also describe polynucleotides associated with stress tolerance and their use in the generation of stress-tolerant plants. US Patent Nos. 5,296,462 and 5,356,816 describe the transformation of plants with polynucleotides encoding proteins involved in cold adaptation in Arabidopsis thaliana to promote cold tolerance.

5 US Patent No. 6,670,528 describes the transformation of plants with polynucleotides encoding polypeptides that bind to stress-sensitive elements to promote tolerance to abiotic stress.

US Patent No. 6,720,477 describes the transformation of plants with a polynucleotide encoding a stress-related signal transduction protein, capable of increasing the tolerance to abiotic stress of transformed plants.

The US Nos. Series 09/938842 and 10/342224 applications describe genes related to abiotic stress and their use to confer tolerance to abiotic stress on plants.

15 US Application Serial No. 10/231035 describes the overexpression of a molybdenum cofactor sulfurase in plants to increase tolerance to abiotic stress.

WO2004 / 104162 of Evogene Ltd. teaches polynucleotide sequences and methods for using them 20 to increase the tolerance of a plant to abiotic stress and / or increase the biomass of a plant.

WO2007 / 020638 from Evogene Ltd. teaches polynucleotide sequences and methods to use them to increase a plant's tolerance to abiotic stress and / or increase the biomass, vigor and / or performance of a plant.

25 WO2007 / 049275 from Evogene Ltd. teaches isolated polypeptides, polynucleotides that encode them to increase a plant's tolerance to abiotic stress, and / or to increase the biomass, vigor and / or performance of a plant.

30 Additional prior art includes United States Patent Applications Nos. 20060183137A1 A1 and 20030056249A1.

US 2004/0123343 discloses sequences of polynucleotides and transgenic plants.

Summary of the invention

In accordance with one aspect of the present invention a method is provided.

According to some embodiments of the invention, the plant cell is part of a plant.

In accordance with some embodiments of the invention, abiotic stress is selected from the group consisting of salinity, drought, lack of water, low temperature, high temperature, heavy metal toxicity, anaerobiosis, nutrient deficit, excess nutrients, pollution atmospheric and UV radiation.

According to some embodiments of the invention, the method further comprises cultivating the plant that expresses the exogenous polynucleotide under conditions of abiotic stress.

Unless otherwise defined, all scientific and / or technical terms used in this document have the same meaning as normally understood by a person skilled in the art to which the

50 invention. Although methods and materials similar or equivalent to those described herein may be used in practice or in the tests of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification will be controlled, including the definitions. In addition, the materials, methods and examples are illustrative only and are not necessarily intended to be limiting.

55 Brief description of the drawings

Some embodiments of the invention are described herein, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is emphasized that

The particularities shown are by way of example and in order to comment illustratively on the embodiments of the invention. In this sense, by taking the description together with the drawings, it becomes clear to those skilled in the art how the embodiments of the invention can be practiced.

In the drawings: 65

FIG. 1 is a schematic illustration of the binary plasmid pGI used to express the isolated polynucleotide sequences of the invention. LD - right limit of T-DNA; LI - left border of T-DNA; H- HindIII restriction enzyme; X - restriction enzyme XbaI; B - BamHI restriction enzyme; S - SalI restriction enzyme; Sm - SmaI restriction enzyme; R-I - EcoRI restriction enzyme; Sc -SacI / SstI / Ecl136II; (numbers) - Length in base pairs; pro NOS = nopaline synthase promoter; NPT-II = neomycin phosphotransferase gene; ter NOS = nopaline synthase terminator; Poly-A signal (polyadenylation signal); GUSintron GUS indicator gene (coding sequence and intron). The isolated polynucleotide sequences of the invention were cloned into the vector while replacing the GUSintron reporter gene. FIGs. 2a-b are images that represent the visualization of the root development of plants grown on transparent plates with agar. The different transgenes were grown on transparent plates with agar for 17 days and the plates were photographed every 2 days beginning on day 7. Figure 2a is an image of a photograph of the plants taken after 12 days on plates with agar. Figure 2b is an image of the root analysis in which the measure of the root length is represented by a red arrow.

Description of specific embodiments of the invention

The present disclosure refers, in some of its aspects, to isolated polypeptides and polynucleotides that encode them, and more particularly, but not exclusively, to methods of using them to increase tolerance to abiotic stress, growth rate, yield, biomass and / or vigor of a plant.

Before explaining in detail at least one embodiment of the disclosure, it should be understood that the invention is not necessarily limited in this application to the details set forth in the following description or illustrated through the Examples. The invention may have other embodiments or be practiced or carried out in various ways.

By limiting the invention to practice, the authors of the present invention have identified new polypeptides and polynucleotides that can be used to increase tolerance to abiotic stress and improve the growth rate, biomass, yield and / or vigor of a plant.

Therefore, as shown later in the Examples section, the authors of the present invention have employed a bioinformatic strategy that combines the clustering and set of database sequences of the genomes of Arabidopsis, rice and other genome plants. of plants available to the public, expressed sequence tags (EST), protein databases and routes and QTL information (Quantitative Trait Locus) with a digital expression profile (“Electronic Northern Blot ”, Northen Electronic Transfer) and have identified polynucleotides and polypeptides that can increase tolerance to abiotic stress and improve growth, biomass, yield and vigor (SEQ ID Nos: 1-200 and 1653 for polynucleotides; SEQ ID Nos: 201-391 and 1655 for polypeptides; Table 1, Example 1). Orthographic assumptions with TEAB (Abiotic Stress Tolerance) of monocot species were identified by aligning ortholog sequences and digital expression profiles (SEQ ID Nos: 392960, 1656-1659 for polynucleotides; SEQ ID Nos: 961-1529, 1660-1663 for the polypeptides; Table 2, Example 1). As further described in Tables 3 and 4 of the Examples section below, representative polynucleotides (polynucleotides of SEQ ID Nos: 1530, 1538, 1532, 1549, 1665, 1566, 1554, 1563, 1557, 1561, 1564 were cloned , 1534, 1536, 1552, 1553, 1666, 1547, 1548, 1556, 1559, 1560, 1654, 1555, 1540, 1543 and 1668). Additional polynucleotides having optimized nucleic acid sequences (polynucleotides of SEQ ID Nos: 1531, 1539, 1533, 1550, 1558, 1562, 1565, 1541, 1667, 1542, 1544, 1537, 1551 and 1545) were prepared. As further described in the Examples section below, transgenic plants were generated that exogenously expressed the cloned and / or optimized polynucleotides of the invention. As shown in Tables 5-76, these plants showed an increase in seedling weight, root coverage, root length and relative growth rate, when grown under conditions of osmotic stress (in the presence of PEG 25%), with nitrogen deficit (in the presence of 0.75 mM nitrogen) or under regular conditions. In addition, as shown in Tables 77-188, the plants that exogenously expressed the polynucleotides of the invention, showed an increase in the area of the rosette, the diameter of the rosette, the average leaf area, the growth rate of the above. , of the biomass of the plants, of the yield of the seeds of the plants, of the weight of 1000 seeds and of the index of harvest, when they were cultivated in conditions of stress by salinity or in normal conditions. Together, these results suggest the use of new polynucleotides and polypeptides of the disclosure to increase tolerance to abiotic stress, and improve the growth rate, biomass, vigor and / or yield of a plant.

Therefore, according to one aspect of the disclosure, a method for increasing tolerance to abiotic stress, growth rate, biomass, yield and / or vigor of a plant is provided. The method is performed by expressing in the plant an exogenous polynucleotide encoding a polypeptide comprising an amino acid sequence that has a homology of at least 60% with the amino acid sequence selected from the group consisting of SEQ ID NO: 201.

The phrase "abiotic stress", as used herein, refers to any adverse effect on the metabolism, growth, reproduction and / or viability of a plant. Therefore, abiotic stress can be induced by suboptimal environmental growth conditions, such as, for example, salinity,

lack of water, water deficit, drought, flood, freezing, low or high temperatures (for example, cooling

or excessive heat), toxic chemical pollution, heavy metal toxicity, anaerobiosis, nutrient deficit, excess nutrients, atmospheric pollution or UV radiation. The implications of abiotic stress are discussed in the Background section.

The phrase "tolerance to abiotic stress" as used herein refers to the ability of a plant to resist abiotic stress without suffering a substantial alteration in metabolism, growth, productivity and / or viability.

As used herein, the phrase "plant biomass" refers to the amount (measured in grams of air-dried or dried tissue) of a tissue produced by the plant in a growing season, which could also determine the, or affect the yield of the plant or the yield per crop area.

As used herein, the phrase "plant yield" refers to the amount (determined by weight, volume or size) or the amount (in numbers) of tissue produced or harvested per plant or per growing season. Therefore, an increased yield could affect the economic benefit that could be obtained from the plant in a given crop area and / or crop time.

As used herein, the phrase "plant vigor" refers to the amount (measured by weight) of tissue that the plant produces in a given time. Therefore, the increase in vigor could determine the, or affect, the yield of the plant or the yield by cultivation time or cultivation area.

As used herein the term "increase" refers to an increase of at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or greater in tolerance to abiotic stress, growth, biomass, yield and / or vigor of a plant compared to a native plant [ie, a plant not modified with the biomolecules (polynucleotides or polypeptides) of the disclosure, for example, an unprocessed plant of the same species that is grown under the same growing conditions].

As used herein, the phrase "exogenous polynucleotide" refers to a heterologous nucleic acid sequence that cannot be expressed naturally in the plant or whose overexpression is desired in the plant. The exogenous polynucleotide can be introduced into the plant in a stable or transient manner, to produce a ribonucleic acid (RNA) molecule and / or a polypeptide molecule. It should be noted that the exogenous polynucleotide may comprise a nucleic acid sequence that is identical or partially homologous to an endogenous nucleic acid sequence of the plant.

As mentioned, the exogenous polynucleotide of the disclosure encodes a polypeptide having an amino acid sequence having a homology of at least about 65%, at least about 65%, at least about 70%, at least about 75%, at less about 80%, at least about 81%, at least about 82%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97% , at least about 98%, at least about 99% or more, that is, 100% with the amino acid sequence selected from the group consisting of SEQ ID NO: 201.

Homology (for example, homology percentage) can be determined using any homology comparison software, including, for example, the National Center or Biotechnology Information (NCBI) BlastP or TBLASTN software, such as using default parameters, when starts from a polypeptide sequence; or the tBLASTX algorithm (available at the NCBI) such as using default parameters, which compares the six conceptual translation products in phase of a nucleotide search sequence (both chains) against a protein sequence database.

Homologous sequences include both orthologous and paralogic sequences. The term "paralogue" refers to gene duplications within the genome of a species that leads to parologous genes. The term "ortholog" refers to homologous genes in different organisms due to an ancestral relationship.

An option to identify orthologs in monocotyledonous plant species is to perform a reciprocal blast search. This can be done through a first blast search that involves applying the blast program to the sequence of interest against any sequence database, such as the NCBI database available to the public that can be found in the: Hypertext Transfer Protocol: // World Wide Web (.) Ncbi (.) Nlm (.)

nih (.) gov. If orthologs are sought in rice, the blast program could be applied to the sequence of interest, for example, against the 28,469 full-length cDNA clones of Oryza sativa Nipponbare, available at the NCBI. Blast results can be filtered. The full length sequences of the filtered or unfiltered results are analyzed again with the blast program (second blast) against the sequences of the organism from which the sequence of interest is derived. Then, the results of the first and second blast are compared. An ortholog is identified when the sequence resulting in the highest score (highest success) in the first blast identifies in the second blast the search sequence (the original sequence of interest) as the best success. Using the same logic, a paralogue (homologue to a gene in the same organism) is found. In the case of large sequence families, the ClustaIW [Hypertext Transfer Protocol: // World Wide Web (.) Ebi (.) Ac (.) Uk / Tools / clustalw2 / index (.) Html] program can be used, followed from a union tree of the nearest neighbor (Hypertext Transfer Protocol: // in (.) Wikipedia (.) org / wiki / Neighbor-joining) that helps to visualize the grouping.

In accordance with some embodiments of the disclosure, the exogenous polynucleotide encodes a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 201.

In accordance with some embodiments of the disclosure the exogenous polynucleotide comprises a nucleic acid sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at less about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96% , at least about 97%, at least about 98%, at least about 99%, for example, 100% identical to the nucleic acid sequence selected from the group q It consists of SEQ ID Nos: 1530, 1531.

Identity (eg, homology percentage) can be determined using any homology comparison software, including, for example, the BlastN computer program of the National Center of Biotechnology Information (NCBI) such as using default parameters.

According to some embodiments of the disclosure, the exogenous polynucleotide is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at less about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% , at least about 99%, for example, 100%, identical to the polynucleotide selected from the group consisting of SEQ ID Nos: 1530, 1531.

In accordance with some embodiments of the disclosure the exogenous polynucleotide is set forth in SEQ ID Nos: 1530, 1531.

As used herein the term "polynucleotide" refers to a single or double stranded nucleic acid sequence that is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a polynucleotide sequence. genomic and / or sequences of composite polynucleotides (for example, a combination of the above).

As used herein, the phrase "complementary polynucleotide sequence" refers to a sequence, which is the result of reverse transcription of messenger RNA using a reverse transcriptase or any other RNA-dependent DNA polymerase. Said sequence can be subsequently amplified in vivo or in vitro using a DNA-dependent DNA polymerase.

As used herein, the phrase "genomic polynucleotide sequence" refers to a sequence (identified or isolated) that comes from a chromosome and therefore represents a contiguous part of a chromosome.

As used herein the phrase "composite polynucleotide sequence" refers to a sequence, which is at least partially complementary and at least partially genomic. A composite sequence may include some exonic sequences necessary to encode the polypeptide of the present invention, as well as some intronic sequences interposed therebetween. Intronic sequences can be from any source, including from other genes, and will typically include conserved splice signaling sequences. Such intronic sequences may additionally include expression regulatory elements that act in cis.

The expression of the nucleic acid sequences encoding the polypeptides of the present invention can be optimized. A non-limiting example of an optimized nucleic acid sequence is provided in SEQ ID NO: 1531, which encodes the polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 201. Examples of such sequence modifications include, but Without limitation, an altered G / C content for a narrower strategy than is typically found in plant species of interest and the withdrawal of atypically found codons in plant species is usually called codon optimization.

The phrase "codon optimization" refers to the selection of DNA nucleotides suitable for use within a structural gene or fragment thereof that addresses the use of codons within the plant of interest. Therefore, an optimized gene or an optimized nucleic acid sequence refers to a gene in which the nucleotide sequence of a native or naturally occurring gene has been modified to use statistically favored or statistically preferred codons in the plant. Typically, the nucleotide sequence is examined at the DNA level and the coding region is optimized for expression in the determined plant species using any suitable method, for example, as described by Sardana et al. (1996, Plant Cell Reports 15: 677-681). In this method, the standard deviation of codon usage, a measure of codon usage bias, can be calculated to first find the proportional quadratic deviation of each codon's use of the native gene with respect to that of highly expressed plant genes, followed of a calculation of the mean square deviation. The formula used is: 1 SDCU = n = 1 N [(Xn - Yn) / Yn] 2 / N, in which Xn refers to the frequency of use of n codons in highly expressed plant genes, in which Yn is the frequency of use of n codons in the gene of interest and N refers to the total number of codons in the gene of interest. Using data from Murray et al. (1989, Nuc Acids Res. 17: 477-498), a Table of the codon usage of highly expressed genes of dicotyledonous plants is compiled.

A method for optimizing the nucleic acid sequence according to the use of preferred codons for a particular plant cell type is based on the direct use, without performing any extra statistical calculation, of Codon Optimization Tables, such as provided online in the Codon Use Database through the NIAS DNA Bank (National Institute of Agrobiological Sciences) in Japan (Hypertext Transfer Protocol: // World Wide Web (.) kazusa (.) or (.) jp / codon /). The codon usage database contains codon usage tables for various different species, each codon usage table having been statistically determined based on the data present in the Genbank.

Using the above Tables to determine the most preferred or most favored codons for each amino acid in a particular species (eg, rice), a naturally occurring nucleotide sequence encoding a protein of interest can be optimized by codons for that particular plant species. . This is done by replacing codons that may have a statistically low frequency in the genome of the particular species with corresponding codons, as regards an amino acid, which are statistically more favored. However, one or more less favored codons can be selected to delete existing restriction sites, to create new sites at possibly useful junctions (5 'and 3' ends to add a signal peptide or termination cassettes, internal sites that could be used to cut and cut and splice segments to each other to produce a correct full length sequence) or to eliminate nucleotide sequences that can negatively affect the stability or expression of mRNA.

The naturally occurring encoded nucleotide sequence may, already in advance of any modification, contain various codons that correspond to a statistically favored codon in a particular plant species. Therefore, codon optimization of the native nucleotide sequence may comprise determining which codons, within the native nucleotide sequence, are not statistically favored with respect to a particular plant, and modifying these codons according to the usage table. of codons of the particular plant to produce a codon-optimized derivative. A modified nucleotide sequence can be completely or partially optimized for the use of plant codons provided that the protein encoded by the modified nucleotide sequence is produced at a higher level than that of the protein encoded by the corresponding native or naturally occurring gene . The construction of synthetic genes by altering the use of codons is described, for example, in PCT Patent Application 93/07278.

Therefore, the disclosure includes nucleic acid sequences described above herein; fragments thereof, sequences that can hybridize with these, sequences homologous to these, sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or replacement of one or more nucleotides, either of origin natural or man-induced, either randomly or in a directed way.

The disclosure provides an isolated polypeptide having an amino acid sequence with a homology of at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least

about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more, for example, 100% with an amino acid sequence selected from the group consisting of SEQ ID NO: 201.

In accordance with some embodiments of the disclosure, the polypeptide is set forth in SEQ ID NO: 201.

The invention also includes fragments of the polypeptides described above and polypeptides having mutations, such as deletions, insertions or substitutions of one or more amino acids, either naturally occurring or man-induced, either randomly or in a directed manner.

The term "plant", as used herein, includes whole plants, ancestors and progeny of plants and parts of the plant, including seeds, shoots, stems, roots (including tubers) and cells, tissues and organs of the plant. plant. The plant can have any shape, including suspension cultures, embryos, meristematic regions, callus tissue, leaves, gametocytes, sporophytes, pollen and microspores. Plants that are particularly useful in the methods of the disclosure include all plants belonging to the super family Viridiplantae, in particular monocotyledonous and dicotyledonous plants that include grass or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp. , Brassica.spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens, Chacoomeles spp., Cinnamomum cassia, Coffea arabica , Coronillia varia, Cotoneaster serotine, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopog on spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Dibeteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehraffine spp., Elehraus, spp. Erythrina spp., Eucalypfus spp., Euclea schimperi, Eulalia villosa, Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingia spp, Freycinetia banksli, Geranium thunbergii, GinAgo biloba, Glycine javanica, Glyphutia spp. Guibourtia coleosperma, Hedysarum spp., Hemaffhia altissima, Heteropogon contoffus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffhelia dissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia, Lethacapus lepuca, Lepedca spu. bainesli, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago saliva, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Ornit hopus spp., Oryza spp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara, Pogona fleckii, Pogonaffhria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp. Rubinia pp. ., Salix spp., Schyzachyrium sanguineum, Sciadopitys vefficillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium trichum. , Tsuga heterophylla, Vaccinium spp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brusela sprouts s, cabbage, rapeseed, carrot, cauliflower, celery, cabbage, flax, kale, lentil, canola, okra, onion, potato, rice, soy, straw, beet, sugar cane, sunflower, tomato, pumpkin, corn, wheat , barley, rye, oats, peanuts, pinto peas, lentils and alfalfa, cotton, soybeans, canola, pepper, sunflower, tobacco, eggplant, eucalyptus, a tree, an ornamental plant, a perennial herb and a forage crop. Alternatively, algae and other plants other than the Viridiplantae superfamily can be used in the methods of the present disclosure.

According to some embodiments of the disclosure, the plants used in the method of the invention are: a crop plant, such as rice, corn, wheat, barley, peanut, potato, sesame, olive, palm oil, banana, soy , sunflower, canola, sugar cane, alfalfa, millet, legumes (beans, pinto peas), flax, lupine, rapeseed, tobacco, poplar and cotton.

Expression of the exogenous polynucleotide of the disclosure in the plant can be effected by transforming one or more plant cells with the exogenous polynucleotide, followed by the generation of a mature plant from the transformed cells and the cultivation of the mature plant under conditions suitable for Exogenous polynucleotide expression in the mature plant.

According to some embodiments of the disclosure, the transformation is effected by introducing into the plant cell a nucleic acid construct that includes the exogenous polynucleotide of some embodiments of the disclosure and at least one promoter capable of directing the transcription of the exogenous polynucleotide into The plant cell. Next, details of appropriate transformation strategies are provided below.

As used herein, the term "promoter" refers to a region of DNA that is located upstream of the transcriptional start site of a gene to which RNA polymerase binds to initiate RNA transcription. The promoter controls where (for example, in which part of a plant) and / or when (for example, at what stage

or state of life of an organism) the gene is expressed.

Any promoter sequence suitable for the construction of the nucleic acid of the present disclosure can be used. According to some embodiments of the disclosure, the promoter is a constitutive promoter, a tissue specific promoter, or an abiotic stress inducible promoter.

Suitable constitutive promoters include, for example, the CaMV 35S promoter (SEQ ID NO: 1546; Odell et al., Nature 313: 810-812, 1985); Arabidopsis At6669 promoter (SEQ ID NO: 1652; see PCT Publication No. WO04081173A2); Ubi 1 of corn (Christensen et al., Plant Sol. Biol. 18: 675-689, 1992); rice actin (McElroy et al., Plant Cell 2: 163-171, 1990); pEMU (Last et al., Theor. Appl. Genet. 81: 581-588, 1991); 19M of the CaMV (Nilsson et al., Physiol. Plant 100: 456-462, 1997); GOS2 (from Pater et al., Plant J Nov; 2 (6): 837-44, 1992); ubiquitin (Christensen et al., Plant Mol. Biol. 18: 675-689, 1992); Rice cyclophilin (Bucholz et al., Plant Mol Biol. 25 (5): 83743, 1994); H3 histone from corn (Lepetit et al., Mol. Gen. Genet. 231: 276-285, 1992); Actin 2 (An et al., Plant J. 10 (1); 107-121, 1996), the CT2 constitutive promoter of the root tip (SEQ ID NO: 1535; see also PCT Application No. IL / 2005 / 000627) and Synthetic Super MAS (Ni et al., The Plant Journal 7: 661-76, 1995). Other constitutive promoters include those indicated in US Pat. Nos. 5,659,026, 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142.

Suitable tissue-specific promoters include, but are not limited to, leaf-specific promoters [such as those described, for example, by Yamamoto et al., Plant J. 12: 255-265, 1997; Kwon et al., Plant Physiol.

105: 357-67, 1994; Yamamoto et al., Plant Cell Physiol. 35: 773-778, 1994; Gotor et al., Plant J. 3: 509-18, 1993; Orozco et al., Plant Mol. Biol. 23: 1129-1138, 1993; and Matsuoka et al., Proc. Natl. Acad. Sci. USA 90: 9586-9590, 1993], preferred seed promoters [eg, of seed-specific genes (Simon, et al., Plant Mol. Biol.

5. 191, 1985; Scofield, et al., J. Biol. Chem. 262: 12202, 1987; Baszczynski, et al., Plant Mol. Biol. 14: 633, 1990), Brazil nut albumin (Pearson 'et al., Plant Mol. Biol. 18: 235-245, 1992), legumin (Ellis, et al. Plant Mol. Biol. 10: 203 -214, 1988), Glutelin (rice) (Takaiwa, et al., Mol. Gen. Genet. 208: 15-22, 1986; Takaiwa, et al., FEBS Letts. 221: 43-47, 1987), Zeína (Matzke et al., Plant Mol Biol, 143) .323-32 1990), napA (Stalberg, et al., Plant 199: 515-519, 1996), Wheat SPA (Albanietal, Plant Cell, 9: 171- 184, 1997), sunflower oleosin (Cummins, et al., Plant Mol. Biol. 19: 873-876, 1992)], specific endosperm promoters [eg, wheat glutelin-1 from BPM and APM (Mol Gen Genet 216: 81-90, 1989; NAR 17: 461-2), gliadins a, wheat byg (EMBO3: 1409-15, 1984), ltrl promoter. of barley, hordeina B1, C, D of barley (Theor Appl Gen 98: 1253-62, 1999; Plant J 4: 343-55, 1993; Mol Gen Genet 250: 750-60, 1996), DOF of Barley (Mena et al., The Plant Journal, 116 (1): 53-62, 1998), Biz2 (EP99106056.7), synthetic promoter (Vicente-Carbajosa et al., Plant J. 13: 629-640, 1998), prolamine NRP33 of rice, Glb-1 rice globulin (Wu et al., Plant Cell Physiology 39 (8) 885-889, 1998), rice alpha-globulin REB / OHP-1 (Nakase et al. Plant Mol. Biol. 33: 513-S22, 1997), ADP-glucose PP of rice (Trans Res 6: 157-68, 1997), ESR gene family of corn (Plant J 12: 235-46, 1997), sorghum gamma-kafirin (PMB 32: 1029-35, 1996)], embryo-specific promoters [eg, rice OSH1 (Sato et al., Proc. Nati. Acad. Sci. USA, 93: 8117-8122), KNOX (Postma- Haarsma et al, Plant Mol. Biol. 39: 257-71, 1999), rice oleosin (Wu et at, J. Biochem., 123: 386, 1998)], and specific flower promoters [eg, AtPRP4, Caleno synthase (chsA) (Van der Meer, et al. , Plant Mol. Biol. 15, 95-109, 1990), LAT52 (Twell et al., Mol. Gen Genet. 217: 240-245; 1989), attach 3].

Suitable abiotic stress inducible promoters include, but are not limited to, salt inducible promoters, such as RD29A (Yamaguchi-Shinozalei et al., Mol. Gen. Genet. 236: 331-340, 1993); drought-inducible promoters, such as the corn rab17 gene promoter (Pla et. al., Plant Mol. Biol. 21: 259-266, 1993), the corn rab28 gene promoter (Busk et. al., Plant J. 11: 1285-1295, 1997) and the promoter of the maize Ivr2 gene (Pelleschi et. Al., Plant Mol. Biol. 39: 373-380, 1999); heat inducible promoters such as the heat inducible hsp80 promoter of tomatoes (US Patent No. 5,187,267).

The nucleic acid construct of some embodiments of the disclosure may additionally include an appropriate selection marker and / or origin of replication. In accordance with some embodiments of the invention, the nucleic acid construct used is a shuttle vector, which can be propagated in both E. coli (where the construct comprises an appropriate selection marker and origin of replication) and is compatible with propagation. in cells The construction according to the present disclosure may be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.

The nucleic acid construct of some embodiments of the disclosure can be used to stably or transiently transform plant cells. In the stable transformation, the exogenous polynucleotide is integrated into the genome of the plant and as such this represents a stable and inherited trait. In transient transformation, the transformed cell expresses the exogenous polynucleotide but is not integrated into the genome and as such this represents a transient trait.

There are various methods for introducing foreign genes into both monocot and dicot plants (Potrykus, I., Annu. Rev. Plant. Physiol., Plant. Mol. Biol. (1991) 42: 205-225; Shimamoto et al., Nature ( 1989) 338: 274-276).

The main methods for effecting the stable integration of exogenous DNA into genomic DNA of plants include two main strategies:

(i)

 Agrobacterium-mediated gene transfer: (1987) Annu. Rev. Plant Physiol. 38: 467-486; Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) pp. 2-25; Gatenby, in Plant Biotechnology, eds Kung, S. and Arntzen, C. J., Butterworth Publishers, Boston, Mass. (1989) p. 93-112.

(ii)

 Direct DNA uptake: Paszkowski et al., in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) pp. 52-68; including methods of direct DNA uptake in protoplasts, Toriyama, K. et al. (1988) Bio / Technology 6: 1072-1074. DNA uptake induced by brief electro-shock of plant cells: Zhang et al. Plant Cell Rep. (1988) 7: 379-384. Fromm et al. Nature (1986) 319: 791-793. Injection of DNA into plant cells or tissues by particle bombardment, Klein et al. Bio / Technology (1988) 6: 559-563; McCabe et al. Bio / Technology (1988) 6: 923-926; Sanford, Physiol. Plant (1990) 79: 206-209; by using micropipette systems: Neuhaus et al., Theor. Appl. Genet (1987) 75: 3 0-36; Neuhaus and Spangenberg, Physiol. Plant (1990) 79: 213-217; transformation with whiskers of silicon carbide or fiberglass from cell cultures, embryo or callus tissues, U.S. Patent No. 5,464,765 or by direct incubation of germinating pollen DNA, DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman, G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p. 197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986)

83: 715-719.

The Agrobacterium system includes the use of plasmid vectors that contain defined segments of DNA that are integrated into the genomic DNA of the plant. The methods of inoculation of plant tissue vary depending on the species of the plant and the Agrobacterium delivery system. A widely used strategy is the foliar disc procedure that can be performed with any tissue explant that provides a good source to initiate differentiation of the entire plant. See, for example, Horsch et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A complementary strategy employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially viable in the creation of transgenic dicotyledonous plants.

There are various methods of direct DNA transfer in plant cells. In electroporation, protoplasts are briefly exposed to a strong electric field. In microinjection, DNA is mechanically injected directly into the cells using very small micropipettes. In the bombardment with microparticles, the DNA is adsorbed into microprojectiles, such as manganese sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated in the cells or tissues of the plant.

After the stable transformation the propagation of the plant is carried out. The most common method to propagate the plant is by seeds. However, regeneration through seed propagation has the defect that, due to heterozygosity, there is an absence of uniformity in the crop, since the seeds are produced by plants according to genetic variations governed by Mendel's laws. Basically, each seed is genetically different and each one will grow with its own specific features. Therefore, it is preferred that the transformed plant is produced in such a way that the regenerated plant has the same features and characteristics as those of the parental transgenic plant. For this reason it is preferred that the transformed plant be regenerated by micropropagation which provides a uniform and rapid reproduction of the transformed plants.

Micropropagation is a process of growing new generations of plants from a single piece of tissue that has been extracted from a selected plant or variety of parental cultivation. This process allows mass reproduction of plants that have the preferred tissue that expresses the fusion protein. The new generation plants that are produced are genetically identical to, and have all the characteristics of, the original plant. The micropropagation allows the mass production of quality plant material in a short period of time and offers a rapid multiplication of the selected crop varieties in the conservation of the characteristics of the original transformed or transgenic plant. The advantages of plant cloning are the speed of plant multiplication and the quality and uniformity of the plants produced.

Micropropagation is a multi-phase procedure that requires the alteration of the culture medium or the culture conditions between the phases. Therefore, the micropropagation process involves four basic phases: phase one, initial tissue culture; phase two, multiplication of tissue culture; phase three, differentiation and formation of the plant; and phase four, cultivation and strengthening in the greenhouse. During phase one of initial tissue culture, tissue culture is established and verified to be free of contaminants. During phase two, the initial tissue culture is multiplied to produce a sufficient number of tissue samples to achieve production objectives. During phase three, tissue samples cultured in phase two are divided and developed into individual seedlings. In phase four, the transformed seedlings are transferred to a greenhouse to be strengthened, where the tolerance of plants to light is gradually increased so that they can grow in a natural environment.

According to some embodiments of the disclosure, transgenic plants are generated by transient transformation of foliar cells, meristematic cells or the entire plant.

Transient transformation can be effected by any of the direct DNA transfer methods described above or by viral infection using modified plant viruses.

Viruses that have been shown to be useful for the transformation of plant hosts include CaMV, Tobacco mosaic virus (TMV), bromine mosaic virus (BMV) and Common Bean Mosaic Virus (BV

or BCMV). The transformation of plants using plant viruses is described in US Pat. No.

4,855,237 (yellow bean mosaic virus; BGV), EP-A 67,553 (TMV), Japanese Published Application No. 6314693 (TMV), EPA 194.809 (BV), EPA 278.667 (BV); and Gluzman, Y. et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, p. 172-189 (1988). WO 87/06261 describes Pseudovirus particles for use in the expression of exogenous DNA in many hosts, including plants.

According to some embodiments of the disclosure, the virus used for transient transformations is an avirulent virus and therefore unable to cause serious symptoms such as reduced growth rate, mosaic, ring spots, leaf curl, yellowing, streaking, formation of pustules, tumor formation and bites. A suitable avirulent virus can be a naturally occurring avirulent virus or an artificially attenuated virus. Virus attenuation can be performed using methods well known in the art including, but not limited to, sublethal heating, chemical treatment or by directed mutagenesis techniques, such as those described, for example, by Kurihara and Watanabe (Molecular Plant Pathology 4: 259- 269, 2003), Galon et al. (1992), Atreya et al. (1992) and Huet et al. (1994).

Suitable virus strains can be obtained from available sources such as, for example, the American Type Culture Collection (ATCC) or by isolation of infected plants. Virus isolation from infected plant tissues can be performed by techniques well known in the art, such as those described, for example, by Foster and Tatlor, Eds. "Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol 81)", Humana Press, 1998. In summary, the tissues of an infected plant, thought to contain a high concentration of A suitable virus, preferably leaves and petals of young flowers, are grown in a buffer solution (eg, phosphate buffer solution) to produce a virus-infected sap that can be used in subsequent inoculations.

The construction of plant RNA viruses for the introduction and expression of non-viral exogenous polynucleotide sequences in plants is demonstrated in the previous references, as well as in Dawson, W. O. et al., Virology (1989) 172: 285-292; Takamatsu et al. EMBO J. (1987) 6: 307-311; French et al. Science (1986) 231: 12941297; Takamatsu et al. FEBS Letters (1990) 269: 73-76; and in U.S. Patent No. 5,316,931.

When the virus is a DNA virus, appropriate modifications can be made to the virus itself. Alternatively, the virus can first be cloned into a bacterial plasmid to facilitate the construction of the desired viral vector with the exogenous DNA. Then, the virus can be extracted from the plasmid. If the virus is a DNA virus, a bacterial replication origin can be attached to the viral DNA, which then replicates the bacteria. Transcription and translation of this DNA will produce the coating protein that will encapsidate the viral DNA. If the virus is an RNA virus, the virus is generally cloned as a cDNA and inserted into a plasmid. Then, the plasmid is used to make all the constructions. The RNA virus is then produced by transcribing the plasmid viral sequence and translating the viral genes to produce one or more coating proteins that encapsulate the viral RNA.

In one embodiment, a plant viral polynucleotide is provided in which the coding sequence of the native coating protein that has been deleted from a viral polynucleotide, a coding sequence of the viral coating protein of a non-native plant and is inserted a non-native promoter, preferably the subgenomic promoter of the non-native coating protein coding sequence, capable of expression in the host plant, packaging the viral polynucleotide of the recombinant plant and guaranteeing a systemic infection of the host by the viral polynucleotide of the recombinant plant. Alternatively, the coating protein gene can be inactivated by insertion of the non-native polynucleotide sequence therein, such that a protein is produced. The viral polynucleotide of the recombinant plant may contain one or more additional non-native subgenomic promoters. Each non-native subgenomic promoter can transcribe or express adjacent genes or polynucleotide sequences in the host plant and cannot recombine with each other or with native subgenomic promoters. Non-native (exogenous) polynucleotide sequences can be inserted adjacent to the native subgenomic viral promoter of the native or non-native viral subgenomic promoters if more than one polynucleotide sequence is included. Non-native polynucleotide sequences are transcribed or expressed in the host plant under the control of the subgenomic promoter to produce the desired products.

In a second embodiment, as in the first embodiment, a recombinant plant viral polynucleotide is provided, except that the coding sequence of the native coat protein is placed adjacent to one of the non-native coat protein sugbenomic promoters in instead of a coding sequence of the non-native coating protein.

In a third embodiment, a recombinant plant viral polynucleotide is provided in which the native coat protein gene is adjacent to its subgenomic promoter and one or more non-native subgenomic promoters have been inserted into the viral polynucleotide. The inserted non-native subgenomic promoters can transcribe or express adjacent genes in a host plant and cannot recombine with each other or with native subgenomic promoters. Non-native polynucleotide sequences can be inserted adjacent to viral promoters of non-native subgenomic plants such that the sequences are transcribed or expressed in the host plant under the control of subgenomic promoters to produce the desired product.

In a fourth embodiment, as in the third embodiment, a recombinant plant viral polynucleotide is provided, except that the coding sequence of the native coating protein is replaced by a coding sequence of the non-native coating protein.

Viral vectors are encapsidated by the coating proteins encoded by the viral polynucleotide of the recombinant plant to produce a recombinant plant virus. The recombinant plant viral polynucleotide

or recombinant plant virus is used to infect appropriate host plants. The recombinant plant viral polynucleotide can replicate in the host, propagate systemically in the host and transcribe or express one or more foreign genes (exogenous polynucleotide) in the host to produce the desired protein.

In Foster and Taylor, eds. "Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol 81)", Humana Press, 1998; Maramorosh and Koprowski, eds. “Methods in Virology” 7 vols, Academic Press, New York 1967-1984; Hill, S.A. "Methods in Plant Virology", Blackwell, Oxford, 1984; Walkey, D.G.A. "Applied Plant Virology", Wiley, New York, 1985; and Kado and Agrawa, eds. "Principles and Techniques in Plant Virology", Van Nostrand-Reinhold, New York can find techniques for virus inoculation in plants.

In addition to the above, the polynucleotide of the present invention can also be introduced into a chloroplast genome whereby the expression of the chloroplast is facilitated.

A technique for introducing exogenous polynucleotide sequences into the chloroplast genome is known. This technique involves the following procedures. First, the plant cells are chemically treated to reduce the number of chloroplasts per cell to approximately one. Thereafter, the exogenous polynucleotide is introduced by bombarding particles in the cells in order to introduce at least one exogenous polynucleotide molecule into the chloroplast. The exogenous polynucleotide is selected in such a way that it can be integrated into the chloroplast genome by homologous recombination easily carried out by enzymes intrinsic to the chloroplast. For this purpose, the exogenous polynucleotide includes, in addition to a gene of interest, at least one polynucleotide stretch that comes from the chloroplast genome. In addition, the exogenous polynucleotide includes a selection marker, which performs sequential selection procedures to confirm that all, or substantially all, copies of the chloroplast genomes, after such selection, will include the exogenous polynucleotide. Additional details regarding this technique are found in US Patent Nos. 4,945,050; Y

5,693,507. A polypeptide can therefore be produced by the chloroplast protein expression system and become integrated into the inner membrane of the chloroplast.

Since tolerance to abiotic stress, growth, biomass, yield and / or vigor in plants may involve the action of multiple genes additively or in synergy (see, for example, Quesda et al., Plant Physiol 130: 951-063, 2002), the present disclosure also contemplates expressing a plurality of exogenous polynucleotides in a single host plant in order to obtain a superior effect on tolerance to abiotic stress, growth, biomass, yield and / or vigor.

The expression of a plurality of exogenous polynucleotides in a single host plant can be accomplished by co-introducing multiple nucleic acid constructs, each including a different exogenous polynucleotide, into a single plant cell. The transformed cell can then be regenerated in a mature plant using the methods described hereinabove.

Alternatively, the expression of a plurality of exogenous polynucleotides in a single host plant can be accomplished by co-introducing into a single plant cell a single nucleic acid construct that includes a plurality of different exogenous polynucleotides. Such a construct can be designed with a single promoter sequence that can transcribe a polycistronic messenger RNA including all different sequences of different exogenous polynucleotides. To allow co-translation of the different polypeptides encoded by the polycistronic messenger RNA, the polynucleotide sequences can be interconnected by an internal ribosome entry site (IRES) sequence that facilitates the translation of the polynucleotide sequences located downstream of the IRES sequence In this case, a transcribed polycistronic RNA molecule encoding the different polypeptides described above will be translated from the 5 ′ protected end and the two internal IRES sequences of the polycistronic RNA molecule to thereby produce in the cell all different polypeptides. Alternatively, the construction may include various promoter sequences, each linked to a different polynucleotide sequence.

The plant cell transformed with the construction including a plurality of different exogenous polynucleotides can be regenerated in a mature plant, using the methods described hereinbefore.

Alternatively, the expression of a plurality of exogenous polynucleotides can be accomplished by introducing different nucleic acid constructs, including different exogenous polynucleotides, into a plurality of plants. Then, the regenerated transformed plants can be crossed and the resulting progeny can be selected with respect to superior traits of tolerance to abiotic stress, growth, biomass, yield and / or vigor, using conventional plant cultivation techniques.

According to some embodiments of the disclosure, the plant that expresses the exogenous polynucleotide (or polynucleotides) is grown under normal conditions.

According to some embodiments of the disclosure, the method further comprises cultivating the plant that expresses the exogenous polynucleotide (or polynucleotides) with abiotic stress.

Thus, the invention includes plants that express exogenously (as described above), the polynucleotide (or polynucleotides) and / or the polypeptide (or polypeptides) of the invention. Once expressed in the plant cell or throughout the plant, the level of the polypeptide encoded by the exogenous polynucleotide can be determined by methods well known in the art such as activity tests, Western blots using antibodies capable of specifically binding to the polypeptide, Enzyme-linked Immunoabsorbent Assay (ELISA), radioimmunoassay (RIA), immunohistochemistry, immunocytochemistry, immunofluorescence and the like.

Methods for determining at the plant the level of transcribed RNA from the exogenous polynucleotide are well known in the art and include, for example, Northen transfer analysis, reverse transcription polymerase chain reaction (RT-PCR) analysis (including Quantitative, semi-quantitative or real-time RT-PCR) and RNA hybridization in situ.

The polynucleotides and polypeptides described hereinbefore can be used in a wide range of economical plants, in a safe and cost-effective manner.

In effect of the transgene (the exogenous polynucleotide encoding the polypeptide) on tolerance to abiotic stress, growth, biomass, yield and / or vigor can be determined using known methods.

Abiotic stress tolerance - Transformed (i.e., expressing the transgene) and non-transformed (natural type) plants are exposed to a condition of abiotic stress, such as lack of water, suboptimal temperature (low temperature, high temperature), deficit of nutrients, excess nutrients, to a condition of saline stress, osmotic stress, heavy metal toxicity, anaerobiosis, atmospheric pollution and UV radiation.

Salinity tolerance test - Transgenic plants with high salt tolerance tolerance are expected to have better germination, vigor or seed growth under conditions with high salt content. Saline stress can be effected in many ways, such as, for example, watering the plants with a hyper osmotic solution, cultivating the plants hydroponically in a hyper osmotic culture solution (for example, Hoagland solution with added salt) or cultivating the plants in a hyper osmotic culture medium [eg, Murashige-Skoog medium (MS medium) at 50% with added salt]. Since the different plants vary considerably in their tolerance with respect to salinity, the saline concentration in the irrigation water, in the culture solution or in the culture medium, can be adjusted according to the specific characteristics of the variety of crop or specific variety of a plant, to infringe a mild or moderate effect on the physiology and / or morphology of the plants (for an orientation on the appropriate concentration see, Bernstein and Kafkafi, Root Growth Under Salinity Stress In: Plant Roots, The Hidden Half 3rd ed. Waisel Y, Eshel A and Kafkafi U. (editors) Marcel Dekker Inc., New York, 2002, and references therein).

For example, a salinity tolerance test can be performed by watering plants at different stages of development with increasing concentrations of sodium chloride (for example 50 mM NaCl, 100 mM, 200 mM, 400 mM) applied from below and from above to guarantee homogeneous salt dispersion. After exposure to the stress condition, plants are frequently monitored until substantial physiological and / or morphological effects appear in wild-type plants. Therefore, the external phenotypic aspect, the degree of wilting and the overall success to reach maturity and produce offspring is compared between the control and transgenic plants. The average quantitative tolerance parameters include, but are not limited to, average humidity and dry weight, growth rate, leaf size, leaf coverage (total leaf area), the weight of the seeds produced, the average seed size and the number of seeds. seeds produced per plant. Transformed plants that do not have substantial physiological and / or morphological effects, or that have higher biomass compared to wild-type plants, are identified as plants tolerant to abiotic stress.

Osmotic tolerance test - Osmotic stress tests (including tests with sodium chloride and PEG) are performed to determine if an osmotic stress phenotype is specific to sodium chloride or if it is a phenotype

General related to osmotic stress. Plants that are tolerant to osmotic stress may have more tolerance to drought and / or freezing. For saline and osmotic stress experiments, the medium is supplemented, for example, with 50 mM NaCl, 100 mM, 200 mM or 15% PEG, 20% or 25%. See also Examples 6 and 7 of the Examples section below.

Drought tolerance test / osmotic test - Drought tolerance is performed to identify genes that confer better survival to plants after lack of intense water. To analyze whether the transgenic plants are more drought tolerant, an osmotic stress produced by the non-ionic sorbitol osmolyte can be performed in the medium. The control and transgenic plants germinate and are grown in vegetable agar plates for 4 days, after which they are transferred to plates containing 500 mM sorbitol. The treatment causes growth retardation, then both transgenic and control plants are compared, measuring the weight of the plant (wet and dry), yield and through measured growth rates such as time for flowering.

On the other hand, soil-based drought explorations are carried out with plants that overexpress the polynucleotides detailed above. The seeds of Arabidopsis control plants, or other transgenic plants that overexpress the polypeptide of the disclosure germinate and transfer to pots. Drought stress is obtained after stopping watering, accompanied by placing the pots on absorbent paper to boost the soil drying rate. Transgenic and control plants are compared to each other when most control plants develop severe wilting. The plants are then irrigated again, obtaining a significant fraction of control plants that show severe wilting. Plants are classified by comparing controls for each of two criteria: tolerance to drought conditions and recovery after re-watering (survival).

Cold stress tolerance - One way to analyze cold stress is as follows. Mature plants (25 days of life) are transferred to chambers for 1 or 2 months at a temperature of 4 ° C, with constitutive light. Then the plants are taken back to the greenhouse. Two weeks later, the damages of the cooling period, resulting from stunting and other phenotypes, are compared between the control and transgenic plants, measuring the weight of the plant (wet and dry), and comparing the measured growth rates such as flowering time, plant size, yield and the like.

Heat stress tolerance - One way to measure heat stress tolerance is to expose plants at temperatures above 34 ° C for a certain period of time. The tolerance of the plant is examined after transferring the plants back to 22 ° C for recovery and evaluation after 5 days with respect to internal controls (non-transgenic plants) or plants not exposed to either cold stress or heat stress .

Germination tests - Germination tests compare the percentage of seeds of transgenic plants that can complete the germination process with respect to the percentage of seeds of control plants that are treated in the same way. Normal conditions are considered, for example, incubations at 22 ° C with daily cycles of 22 hours of light and 2 hours of darkness. The germination and vigor of the seedling are evaluated between 4 and 14 days after planting. The basal medium is MS medium (Murashige and Skoog, 1962 Plant Physiology 15, 473-497) at 50%.

Germination is also checked under unfavorable conditions such as cold (incubating at temperatures below 10 ° C instead of 22 ° C) or using seed inhibition solutions containing high concentrations of an osmolyte such as sorbitol (at concentrations of 50 mM, 100 mM, 200 mM, 300 mM, 500 mM, and up to 1000 mM) or applying increasing concentrations of salt (50 mM NaCl, 100 mM, 200 mM, 300 mM, 500 mM).

Effect of the transgene on the growth, biomass, yield and / or vigor of the plant - The vigor of the plant can be calculated by increasing the growth parameters such as leaf area, fiber length, Rosette diameter, the fresh weight of the plant and similar by time interval.

The growth rate can be measured using digital analysis of plant growth. For example, images of plants that grow in greenhouses in plots can be captured every three days and the rosette area can be calculated by digital analysis. The rosette area growth is calculated using the difference of the rosette area between sampling days divided by the difference in days between samples.

Seed yield measurements can be made by collecting the total seeds of 8-16 plants together, weighing them using an analytical balance and dividing the total weight by the number of plants. The growth area per seed can be calculated in the same way although taking into account the determined growth area of a single plant. The increase in seed yield per growth area can be obtained by increasing the seed yield per plant and / or increasing the number of plants capable of growing in a given area.

The evaluation of the seed yield per plant can be done by measuring the quantity (weight or size) or number (that is, number) of the dried seeds produced and harvested from 8-16 plants and divided by the number of plants.

The evaluation of the growth rate can be done by measuring the biomass of the plant produced, the area of the rosette, the leaf size or the root length by time (which can be measured in cm2 per day of leaf area).

Fiber length can be measured using a fibrograph. The fibrograph system was used to count the length in terms of "Average of the Upper Half" length. The average length of the upper half (MMS) is the average length of the longest half of the fiber distribution. The fibrograph measures length in lengths by sections at a certain percentage point (Hypertext Transfer Protocol: // World Wide Web (.) Cottoninc (.) Com / ClassificationofCotton /? Pg = 4 # Length).

Therefore, the present invention is of high agricultural value to promote the yield of commercially desired crops (eg, biomass of vegetative organs such as poplar wood, or reproductive organs such as various seeds or seed biomass).

As used herein, the term "approximately" refers to 10%.

The terms "comprises", "comprising", "including", "including" and "having" and their conjugates mean "including but not limited to".

The expression "consisting of" means "including and limited to".

The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, stages and / or parts, but only if the ingredients, stages and / or additional parts do not materially alter the basic and new characteristics of the composition, method or structure claimed.

As used herein, the singular form "one", "one", "the" and "the" include plural references unless the context clearly dictates otherwise. For example, the expression "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of the present invention may be presented in an interval format. It should be understood that the description in interval format is solely for convenience and brevity and should not be considered as an inflexible limitation of the scope of the invention. Therefore, it should be considered that in the description of an interval all possible subintervals have been specifically disclosed as well as the individual numerical values within the interval. For example, the description of an interval, such as 1 to 6, should be considered to have been specifically disclosed in subintervals such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, from 3 to 6, etc., as well as in individual numbers within that range, for example, 1, 2, 3, 4, 5 and 6. This applies regardless of the amplitude of the interval.

Provided that a numerical range is indicated herein, it is understood to include any number (fraction or integer) cited within the indicated range. The phrases "which varies / varies between" a first indicated number and a second indicated number and "which varies / varies from" a first indicated number "to" a second indicated number, are used interchangeably herein and are intended to include the first and second indicated number and all numbers, fractions or integers, among them.

As used herein, the term "method" refers to ways, means, techniques and procedures to perform a given task including, but not limited to, those ways, means, techniques and procedures that are well known, or easily developed in ways. , means, techniques and procedures known to the chemical, pharmacological, biological, biochemical and medical technicians.

It is appreciated that certain features of the invention, which for clarity are described in the context of different embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the invention, which are briefly described in the context of a single embodiment, can also be provided separately or in any suitable sub-combination or, as appropriate, in any described embodiment other than the invention. Certain characteristics described in the context of the various embodiments should not be considered essential characteristics of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention have been defined above herein and, as claimed later in the claims section, find experimental support in the following examples.

Examples

Now, reference is made to the following examples, which together with the above description illustrate some embodiments of the invention in a non-limiting manner. Generally, the nomenclature used herein and in the laboratory procedures used in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. These techniques are explained in detail in the bibliography. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies such as those set forth in US Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); Available immunoassays are described extensively in the patent and scientific literature, see, for example, US Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 AND 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996). Other general references are provided throughout this document. It is believed that the procedures herein are well known in the art and are provided for convenience to the reader.

EXAMPLE 1

IDENTIFICATION OF GENE ASSUMPTIONS THAT INCREASE TOLERANCE TO ABIOTIC STRESS AND OR PERFORMANCE / BIOMASS

The authors of the present invention have identified genes that increase abiotic stress tolerance (TEAB) and / or the growth / yield / biomass / vigor rate, as follows. The genes were validated in vivo as previously described in WO2004 / 104162 this assignee. All nucleotide sequence data sets used herein originated from publicly available databases. Sequence data from 50 different species (mainly plant species) were entered into a single comprehensive database.

Other information on gene expression, protein annotation, enzymes and pathways is also incorporated. The main databases used include:

●

Genomes

○

 Arabidopsis genome [TAIR genome version 6 (Hypertext Transfer Protocol: // World Wide Web (.) Arabidopsis (.) Org /)]

○

 Rice genome [construction IRGSP 4.0 (Hypertext Transfer Protocol: // rgp (.) Dna (.) Affrc (.) Go (.) Jp / IRGSP /)].

○

 Poplar [Populus trichocarpa version 1.1 of JGI (assembly version v1.0) (Hypertext Transfer Protocol: // World Wide Web (.) Genome (.) Jgi-psf (.) Org /)]

○

Brachypodium [JGI 4x Assembly Hypertext Transfer Protocol: // World Wide Web (.) Brachpodium (.) Org)]

○

 Soy [DOE-JGI SCP, version Glyma0 (Hypertext Transfer Protocol: // World Wide Web (.) Phytozome (.) Net /)]

○

 Grape [NCBI WGS assembly ftp: // ftp (.) Ncbi (.) Nih (.) Gov / genbank / wgs /)]

○

 Ricino [TIGR / J Craig Venter Institute 4x assembly

○

 Hypertext Transfer Protocol: // msc (.) Jcvi (.) Org / r_communis

○

 Sorghum [DOE-JGI SCP, version Sbi1 Hypertext Transfer Protocol: // World Wide Web (.) Phytozome (.) Net /)].

●

 The sequences of EST (from the acronym Expressed Sequence Tags, expressed sequence tags) and mRNA were extracted from

○

 GeneBank versions 154, 157, 160, 161, 164, and 165 (Hypertext Transfer Protocol: // World Wide Web (.) Ncbi (.) Nlm (.) Nih (.) Gov / dbEST /)

○

 RefSeq (Hypertext Transfer Protocol: // World Wide Web (.) Ncbi (.) Nlm (.) Nih (.) Gov / RefSeq /).

○

TAIR (Hypertext Transfer Protocol: // World Wide Web (.) Arabidopsis (.) Org /).

●

 Protein databases and routes were extracted from

○

 Uniprot (Hypertext Transfer Protocol: // World Wide Web.expasy.uniprot.org/).

○

 AraCyc (Hypertext Transfer Protocol: // World Wide Web (.) Arabidopsis (.) Org / biocyc / index (.) Jsp).

○

 ENZYME (Hypertext Transfer Protocol: //expasy.org/enzyme/).

●

The microarray data sets were downloaded from

○

 GEO (Hypertext Transfer Protocol: // World Wide Web.ncbi.nlm.nih.gov/geo/)

○

TAIR (Hypertext Transfer Protocol: // World Wide Web.arabidopsis.org/).

○

 Evogene patent cotton fiber microarray data

●

QTL information (acronym for Quantitative Trait Locus, quantitative locus

○ Gramene (Hypertext Transfer Protocol: // World Wide Web (.) Gramene (.) Org / qtl /).

Databases were assembled to build a broad, rich, reliable, annotated and easy-to-analyze database comprising genomic mRNA, EST and DNA sequence data from various cultures as well as gene expression data, annotation of proteins and routes, QTL, and other relevant information.

The assembly of the databases included an application of gene refinement, structuring, annotation and analysis tools that allowed the construction of a customized database for each gene discovery project. The tools of gene refinement and structuring make it possible to reliably detect splice variants and antisense transcripts, generating an understanding of the possible various phenotypic results of a single gene. The ability of the “LEADS” platform of Compugen LTD to analyze the human genome has been confirmed and accepted by the scientific committee (“Widespread Antisense Transcription”, Yelin, et al. (2003) Nature Biotechnology 21, 379-85; “Splicing of Alu Sequences ”, Lev-Maor, et al. (2003) Science 300 (5623), 1288-91), and has also proven to be the most effective in plant genomics.

EST grouping and gene assembly - The “genomic LEADS” version was used to group and assemble arabidopsis and rice genes. This tool allows more accurate grouping of EST and mRNA sequences in the genome and predicts gene structure as well as alternative splicing events and antisense transcription.

For organisms with complete genome sequence data not available, “expressed LEADS” was applied, as well as the TIGR (Hypertext Transfer Protocol: // World Wide Web (.) Tigr (.) Org /) computer program. The results of the two grouping tools were compared and in the cases in which the groups provided by the two tools were significantly different, the two versions were presented and considered.

Gene annotation - the expected genes and proteins (as) were noted as follows:

•

We searched with Blast (Hypertext Transfer Protocol: // World Wide Web (.) Ncbi (.) Nlm (.) Nih (.) Gov (.) Library (.) Vu (.) Edu (.) Au / BLAST /) against all plant UniProt sequences (Hypertext Transfer Protocol: // World Wide Web (.) Expasy (.) Uniprot (.) Org /).

•

Calculations were used with Frame-Finder (Hypertext Transfer Protocol: // World Wide Web (.) Ebi (.) Ac (.) Uk / ~ guy / estate /) with default statistical values to predict protein sequences for each transcribed

•

The expected proteins were analyzed with InterPro (Hypertext Transfer Protocol: // World Wide Web (.) Ebi (.) Ac (.) Uk / interpro /).

•

Anti-Blast proteins from the AraCyc and ENZYME databases were used to map the transcripts intended for the AraCyc routes.

•

Each transcript was compared using the tblastx (Hypertext Transfer Protocol: // World Wide Web (.) Ncbi (.) Nlm (.) Nih (.) Gov (.) Library (.) Vu (.) Edu (. ) au / BLAST /) against the rest of the databases of other organisms to validate the accuracy of the expected protein sequence and for an effective ortholog detection.

Gene expression profile - Very few data sources were used to perform the gene expression profile, particularly microarray and digital expression profile data (see below). According to the gene expression profile, a correlation analysis was performed to identify genes that were co-regulated in different stages of development and environmental conditions.

Microarray data sets available to the public from the TAIR and NCBI GEO sites were downloaded, renormalized and integrated into the databases. The expression profile was one of the most important resource data to identify genes important for TEAB. In addition, when homologous genes from different cultures were sensitive to TEAB, the genes were marked as "highly predictive of improving TEAB."

A summary of the digital expression profile was compiled for each group according to all the keywords included in the sequence records that comprised the group. Digital expression, also known as Northern Electronic transfer, is a tool that shows a virtual expression profile based on the EST sequences that make up the gene group. The tool can provide the expression profile of a group in terms of the anatomy of the plant (the tissues / organs being the expressed gene), development phase (the development phases to which a gene can be found) and the profile of treatment (which provides the physiological conditions in which a gene is expressed, such as drought, cold, pathogen infection, etc.). Given a random distribution of ESTs in different groups, the digital expression provides a probability value that describes the probability of a group that has a total of N ESTs to contain X ESTs from a given library collection. To carry out the probability calculations, the following are taken into account: a) the number of ESTs in the group, b) the number of a EST of the libraries involved and related, c) the total number of ESTs available that represent the species. Therefore, groups with low probability values are greatly enriched with EST from the group of libraries of interest indicating a specialized expression.

The concepts of orthology and paralogy have recently been applied to functional characterizations and classifications on the scale of comparisons of the entire genome. Orthologs and paralogs constitute two main types of homologues, the first evolved from a common ancestor by specialization and the latter are related by duplication events. Paralogs are supposed to arise from ancient duplication events whose function has probably diverged while true orthologs are more likely not to retain the same function throughout evolution.

To investigate and identify additionally orthologous genes of monocot species with TEAB, two computer methods were integrated:

(i)

Method for aligning orthologous sequences - based on the construction of orthologous groups across multiple eukaryotic taxa, using modifications in the Markov group algorithm to group orthologous and paralogical assumptions. These supposed orthologs were further organized into a phylogram - a diagram with ramifications (tree) that is supposed to be an estimate of a phylogeny of genes.

(ii)

 Method for generating "Digital Expression" gene expression profiles - the authors of the present invention have made considerable effort directed to annotation sequences. Expression data were analyzed and EST libraries were classified using a fixed vocabulary of common terms such as experimental treatments. The annotations of all the ESTs grouped for a gene were statistically analyzed by comparing their frequency in the group against their abundance in the database, which allowed to construct a profile of number and graph expression of that gene, which is called “digital expression "

The logic of using these two complementary methods is based on the assumption that true orthologs probably retain the same function throughout evolution. These two methods (sequence and expression pattern) provide two different sets of indications depending on the similarities between two homologous genes, similarities in the sequence level - same amino acids in the protein domains and similarities in the expression profiles.

In total, 110 genes were identified that had a major impact on TEAB when they were overexpressed in plants. The identified TEAB genes, their cured polynucleotide and polypeptide sequences, as well as their updated sequences according to the GenBank database are summarized below in Table 1 of this document.

Polynucleotides and polypeptides that have significant homology with the identified TEAB genes have been identified from the databases using the BLAST software with the BlastX algorithm. The search nucleotide sequences were those of SEQ ID Nos: 1, 3, 5, 7, 9, 10, 11, 13, 15, 16, 17, 19, 21, 23, 25, 26, 28, 29, 30, 32, 34, 36, 37, 38, 40, 42, 44, 46, 48, 50, 52, 54, 55, 57, 59, 61, 63, 65, 67, 69, 71.73, 75, 77, 79, 81, 82, 84, 86, 88, 90, 91, 93, 94, 96, 98, 100, 101, 103, 105, 107, 109, 111, 113, 115, 116, 118, 119, 121, 122, 124, 126, 128, 130, 132, 134, 135, 138, 140, 142, 143, 145, 147, 149, 151, 153, 155, 157, 161, 163, 165, 168, 169, 170, 171, 173, 175, 177, 179, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198 and 1653 and the identified TEAB counterparts are provided in the following Table 2.

EXAMPLE 2

GENERATION OF THE TEAB GENES ASSUMPTIONS

Various DNA sequences of the genes were synthesized with TEAB (Abiotic Stress Tolerance) through the GeneArt (Hypertext Transfer Protocol: // World Wide Web (.) Geneart (.) Com /). Synthetic DNA was designed in silico (with a computer), based on the encoded amino acid sequences of TEAB genes and using codon usage tables calculated from plant transcriptomes (in the codon usage database available online at the Hypertext Transfer Protocol: // World Wide Web (.) kazusa (.) or (.) jp / codon can be found examples of such Tables). The optimized coding sequences were designed without introducing changes in the encoded amino acid sequence although using preferred codons for expression in dicotyledonous plants (mainly tomato and Arabidopsis) and in monocotyledonous plants such as corn. At least one silent mutation was introduced into the sequence by 20 pairs of nucleotides compared to the original sequences to avoid possible silencing when the gene was overexpressed in the target culture. To the optimized sequences were added the following restriction enzymes Sall, Xbal, BamHI, Smal at the 5 'end and Sacl at the 3' end. The sequences synthesized by the provider (GeneArt, Gmbh) were cloned into the plasmid pCR-Script.

EXAMPLE 3

GENE CLONING AND GENERATION OF BINARY VECTORS FOR EXPRESSION IN PLANTS

To validate their role in improving TEAB and performance, the selected genes were overexpressed in plants as follows.

Cloning strategy

The genes selected from those presented in Example 1 were cloned into binary vectors for the generation of transgenic plants. For cloning, full-length Open Reading Frame (ORF) acronym was identified. Groups of ESTs and in some cases mRNA sequences were analyzed to identify the entire open reading phase by comparing the results of various translation algorithms with known proteins from other plant species.

To clone full-length cDNAs, reverse transcription (TR) was performed followed by polymerase chain reaction (PCR; RT-PCR) in total RNA extracted from leaves, roots or other tissues of the plant, which were developed under normal conditions or in conditions with nutrient deficits. Total RNA extraction, cDNA production and PCR amplification were performed using conventional protocols, described in any document (Sambrook J., EF Fritsch, and T. Maniatis. 1989. Molecular Cloning. A Laboratory Manual., 2nd Ed Cold Spring Harbor Laboratory Press, New York.), Known to those skilled in the art. The PCR products were purified using the PCR purification kit (Qiagen).

Normally, 2 sets of primers were prepared for amplification of each gene, by nested PCR (which means expanding the gene first using external primers and then using the PCR product produced as a template for a second PCR reaction, in which the internal set of primers is used). Alternatively, one or two of the internal primers were used for gene amplification, both in the first and in the second PCR reaction (meaning that only 2-3 primers were designed for one gene). To facilitate further cloning of the cDNAs, 8-12 bp of extension was added at the 5 ’end of each internal primer. The primer extension includes an endonuclease restriction site. Restriction sites are selected using two parameters: (a) the restriction site does not exist in the cDNA sequence; and (b) restriction sites in the direct and reverse primers are designed such that the digested cDNA is inserted in the direction in the direction of the binary vector used for the transformation. In the following Table 3, the primers used for the cloning of TEAB genes are provided.

Table 3

TEAB genes cloned from cDNA or genomic DNA libraries and primers used for cloning

Gen Id

Polynucleotide SEQ ID No. of the cloned gene Polypeptide SEQ ID No. of the encoded polypeptide Restriction enzymes used for cloning Primers used for amplification (SEQ ID NO:)

MAB1

1530 201 EcoRV MAB1 EF EcoRV MAB1_NF_EcoRV MAB1_ER_EcoRV MAB1_NR_EcoRV

MAB1_GA (optimized for expression in corn and G. Max)

1531  AND and Synthetic product (from pGA14_MAB1_GA)

MAB14

 1538 219 EcoRV MAB14 EF EcoRV MAB14_NF_EcoRV MAB14_ER_EcoRV MAB14_NR_EcoRV

MAB14_GA (optimized for expression in corn)

1539 Synthetic product (from pGA14_MAB14_GA)

MAB10

 1532 212 Get out, Xbal MAB 10 F Salt -MAB 10_Ext_R Xba -MAB10_NR_Xba

MAB10_GA (optimized for expression in corn)

1533 Synthetic product (from pGA118_MAB10_GA)

TEAB genes cloned from cDNA or genomic DNA libraries and primers used for cloning

Gen Id

Polynucleotide SEQ ID No. of the cloned gene Polypeptide SEQ ID No. of the encoded polypeptide Restriction enzymes used for cloning Primers used for amplification (SEQ ID NO:)

MAB25

1549d 237 PstI, Smal MAB25 EF PstI MAB25_NF_PstI - MAB25_ER_SmaI - MAB25_NR_SmaI -

MAB25 GA (optimized for expression in corn)

1550 Synthetic product (from pGA118_MAB10_GA)

MAB134

 1665 311 Get out, Xbal MAB134_EF_SalI - MAB134_NF_SalI - MAB134_ER_XhaI MAB134_NR_XbaI -

MAB99

1566 283 I left, Scal MAB99_NF_SalI - MAB99_NR_SacI -

MAB36

 1554 254 Get out, Xbal MAB_36 F_Sal - MAB 36 Ext R Xba -MAB 36 NR Xba -

TEAB genes cloned from cDNA or genomic DNA libraries and primers used for cloning

Gen Id

Polynucleotide SEQ ID No. of the cloned gene Polypeptide SEQ ID No. of the encoded polypeptide Restriction enzymes used for cloning Primers used for amplification (SEQ ID NO:)

MAB7

1563 208 Get out, Xbal, MAB 7 ExF Salt - MAB 7 NF Salt -MAB_7 Ex R Xba - MAB_7 NR_Xba -

MAB44

1557 267 I left, Sacl MAB 44 NF salt MAB 44 NR Sc

MAB44_GA (optimized for expression in corn)

1558 Synthetic product (from pCR4Blunt- TOPO_MAB44_GA)

MAB6

1561 207 Get out, Xbal MAB_6 - Ex_F_Sal -MAB_6 NF_Sal - MAB 6 - Ext R_XbaI -MAB_6 - R_XbaI -

MAB6_GA (optimized for expression in corn)

1562 Synthetic product (from pGA15_MAB6_GA)

MAB9

1564 211 EcoRV MAB9_F_EcoRV MAB9_ER_EcoRV MAB9_NR_EcoRV

TEAB genes cloned from cDNA or genomic DNA libraries and primers used for cloning

Gen Id

Polynucleotide SEQ ID No. of the cloned gene Polypeptide SEQ ID No. of the encoded polypeptide Restriction enzymes used for cloning Primers used for amplification (SEQ ID NO:)

MAB9_GA (optimized for expression in corn and G. Max)

1565 Synthetic product (from pGA15_MAB9_GA)

MAB100

 1534 284 Get out, Xbal MAB_100_EF_SalI - MAB100_NF_SalI - MAB100_ER_XbaI - MAB100_NR_XbaI

MAB 13

1536 217 Sacl, Sall MAB13F_SalI_new MAB 13 ExR Sc MAB_13_F_Sal MAB_13_NR_Sc

MAB32

 1552 247 EcoRV MAB32_F_EcoRV - MAB32 ER EcoRV - MAB32_NR_EcoRV -

MAB35

 1553 252 SmaI MAB35_F MAB35_ER_SmaI - MAB35_NR_SmaI -

TEAB genes cloned from cDNA or genomic DNA libraries and primers used for cloning

Gen Id

Polynucleotide SEQ ID No. of the cloned gene Polypeptide SEQ ID No. of the encoded polypeptide Restriction enzymes used for cloning Primers used for amplification (SEQ ID NO:)

MAB146

 1666 334 Get out, Xbal MAB146_F_Sal - MAB146_ER_Xba -MAB146_NR_Xba -

MAB2

1547 Not coded Get out, Xbal MAB2_F_SalI MAB2_R_Xba

MAB20

1548 229 PstI, Smal MAB20_EF_PstI - MAB20_NF_PstI - MAB20_ER_SmaI - MAB20_NR_SmaI -

MAB43

1556 265 PstI, Smal MAB43_NF_PstI MAB43_ER_SmaI MAB43_NR_SmaI

TEAB genes cloned from cDNA or genomic DNA libraries and primers used for cloning

Gen Id

Polynucleotide SEQ ID No. of the cloned gene Polypeptide SEQ ID No. of the encoded polypeptide Restriction enzymes used for cloning Primers used for amplification (SEQ ID NO:)

MAB46

1559 271 I left, Sacl MAB_46_ExF_Sal - MAB_46_NF_Sal - MAB46 ExR Sc MAB_46_NR_Sc -

MAB50

 1560 277 SmaI MAB_50_ExF_Sal MAB50_NF MAB50_ExR_Sac MAB50_NR_Sma

MAB66

1654 1655 Get out, Xbal MAB66_F_Sal - MAB66_ER_Xba - MAB66_NR_Xba -

MAB4

1555 205 EcoRV MAB4_EF_EcoRV - MAB4_NF_EcoRV - MAB4_ER_EcoRV - MAB4_NR_EcoRV -

MAB15_GA (optimized for expression in Arabidopsis and corn)

1541 221 XbaI, Sacl Synthetic product (from pGA4_MAB15)

TEAB genes cloned from cDNA or genomic DNA libraries and primers used for cloning

Gen Id

Polynucleotide SEQ ID No. of the cloned gene Polypeptide SEQ ID No. of the encoded polypeptide Restriction enzymes used for cloning Primers used for amplification (SEQ ID NO:)

MAB15a_G A (optimized for expression in corn)

Synthetic product (from pGA18_ MAB15a_GA)

MAB15_GA _original (original sequence, not optimized)

1540 Synthetic product (from pGA14_MAB15_ (EV0220) -original)

MAB17_GA (optimized for expression in Arabidopsis and corn)

1542 224 XbaI, Sacl Synthetic product (from pGA4_MAB17)

MAB17a_GA (optimized for expression in corn)

1544 Synthetic product (from pCR4Blunt- TOPO_MAB17a_GA)

MAB17_GA _original (original sequence, not optimized)

1543 Synthetic product (pGA14_MAB17_ (EVO222) -original)

MAB137_GA (optimized for expression in corn, Arabidopsis and tomato)

1537 317 XbaI, Sacl Synthetic product (from pGA15_MAB137)

MAB3_GA (optimized for expression in corn, Arabidopsis and tomato)

1551  203 XbaI, Sacl Synthetic product (from pCR4Blunt- Topo_MAB3)

MAB3_GA_ original (original sequence, not optimized)

1688 Synthetic product (from pGA14_MAB3_ (EVO235) -original)

MAB18_GA (optimized for expression in Arabidopsis and corn)

1545 225 XbaI, Sacl Synthetic product (from pGA4_MAB18)

Control Gene: GUI

1664

TEAB genes cloned from cDNA or genomic DNA libraries and primers used for cloning

Gen Id

Polynucleotide SEQ ID No. of the cloned gene Polypeptide SEQ ID No. of the encoded polypeptide Restriction enzymes used for cloning Primers used for amplification (SEQ ID NO:)

Table 3: The cloned TEAB genes and the control gene (or genes) are presented by the ID number of the gene and SEQ ID NO: of the polynucleotide. Primers and restriction enzymes used to clone TEAB genes are also presented.

The PCR products were digested with restriction endonucleases (Roche, Switzerland) according to the sites designed on the primers (Table 3). Each digested PCR product was inserted into a vector with high copy number originating from the plasmid vector pBlue-script KS (plasmid vector pBlue-script KS, Hypertext Transfer Protocol: // World Wide Web (.) Stratagene (.) com / manuals / 212205 (.) pdf). In the case of the vector with high copy number originated from the plasmid vector pBlue-script KS (pGN) the PCR product was inserted into the plasmid with high copy number upstream of the NOS terminator (SEQ ID NO: 1651) originated from the binary vector pBI 101.3 (GenBank Accession No. U12640, nucleotides 4417 to 4693), Table 4 below. In other cases (pKSJ_6669a) the At6669 promoter (SEQ ID NO: 1652) has already been cloned in the pBlue-script KS, so that

10 the gene is introduced downstream of the promoter (Table 4 below).

Sequencing of the inserted genes was performed, using the ABI 377 sequencer (Applied Biosystems). In some cases, after confirming the sequences of the cloned genes, the cloned cDNA accompanied by the NOS terminator was introduced into the binary vectors pGI containing the At6669 promoter by digestion with

15 appropriate restriction endonucleases. In other cases, the cloned cDNA accompanied with the At6660 promoter was introduced into the pGI vector (which no longer had the At6669 promoter. In any case after inserting a single copy of the NOS terminator was made (SEQ ID NO: 1651). digested products and the linearized plasmid vector were ligated using the enzyme T-DNA ligase (Roche, Switzerland).

20 Table 4

Cloned genes from cDNA or genomic DNA libraries in the high copy plasmid

Gene name

Plasmid with high copy number Amplified from

MAB1

pKSJ_6669 RNA

MAB1

Gen art

MAB10

Gen art

MAB10

pGN RNA

MAB14

pKSJ_6669 RNA

MAB14

Gen art

MAB15

pGN Gen Art (3 plasmids)

MAB17

pGN Gen Art (3 plasmids)

MAB137

 pGN Gen art

MAB25

pKSJ_6669 RNA

MAB25

Gen art

MAB3

pGN Gen Art (2 plasmids)

MAB44

 pGN RNA

MAB44

Gen art

MAB6

Gen art

MAB9

pKSJ_6669 RNA

MAB9

Gen art

MAB100

 pGN RNA

MAB13

pGN RNA

MAB134

 pGN RNA

MAB18

pGN Gen

MAB2

pGN RNA

MAB20

pKSJ_6669 RNA

MAB146

 pGN RNA

MAB32

pKSJ_6669 RNA

MAB35

pKS_6669 RNA

MAB36

pGN RNA

MAB43

pKSJ_6669 RNA

MAB46

pGN RNA

MAB50

pKSJ_6669 RNA

MAB7

pGN RNA

MAB99

pGN RNA

Cloned genes from cDNA or genomic DNA libraries in the high copy plasmid

Gene name

Plasmid with high copy number Amplified from

MAB66

pGN RNA

MAB4

pKSJ_6669 RNA

Table 4

The pPI plasmid vector was constructed by inserting a poly- (A) synthetic signal sequence, originating from the basic plasmid vector pGL3 (Promega, GenBank Accession No. U47295; nucleotides 4658-4811) at the HindIII restriction site of the binary vector pBI101.3 (Clontech, GenBank Accession Number U12640). pGI (Figure 1) is similar to pPI, but the original gene in the main chain is GUS-Intron instead of GUS.

At6669, the promoter sequence of Arabidopsis thaliana (set forth in SEQ ID NO: 1652) was inserted into the binary vector pPI, upstream of the cloned genes using the restriction enzymes HindIII and SalI or BamHI (Roche), followed by ligation of DNA and binary plasmid extraction from E. coli positive colonies, as described above.

Positive colonies were identified by PCR using primers that were designed to encompass the introduced promoter (At6669) and the gene cloned into the binary vector. In all cases the direct PCR primer was the primer set forth in SEQ ID NO: 1650 (of the At6669 promoter) and the reverse primers (derived from the specific cloned gene) were the following: for MAB1, the reverse primer was SEQ ID No.: 1570; for MAB14, the reverse primer was SEQ ID NO: 1574, for MAB10, the reverse primer was SEQ ID NO: 1577; for MAB25, the reverse primer was SEQ ID NO: 1581; for MAB134, the reverse primer was SEQ ID NO: 1585; for MAB99, the reverse primer was SEQ ID NO: 1587; for MAB36, the reverse primer was SEQ ID NO: 1590; for MAB7, the reverse primer was SEQ ID NO: 1594; for MAB44, the reverse primer was SEQ ID NO: 1596; for MAB4, the reverse primer was SEQ ID NO: 1600; for MAB9, the reverse primer was SEQ ID NO: 1603 (MAB9); for MAB100, the reverse primer was SEQ ID NO: 1606; for MAB13, the reverse primer was SEQ ID NO: 1611; for MAB32, the reverse primer was SEQ ID NO: 1614; for MAB35, the reverse primer was SEQ ID NO: 1617; for MAB146, the reverse primer was SEQ ID NO: 1620; for MAB2, the reverse primer was SEQ ID NO: 1622; for MAB20, the reverse primer was SEQ ID NO: 1626; for MAB43, the reverse primer was SEQ ID NO: 1629; for MAB46, the reverse primer was SEQ ID NO: 1633; for MAB50, the reverse primer was SEQ ID NO: 1637; for MAB66, the reverse primer was SEQ ID NO: 1640; for MAB4, the reverse primer was SEQ ID NO: 1644; for the synthetic gene MAB15, the reverse primer was SEQ ID NO: 1645; for the synthetic gene MAB17, the reverse primer was SEQ ID NO: 1646; for the synthetic gene MAB18, the reverse primer was SEQ ID NO: 1647; for the synthetic MAG137 gene, the reverse primer was SEQ ID NO: 1648; and for the synthetic MAB3 gene, the reverse primer was SEQ ID NO: 1649, which were designed to encompass the introduced promoter and the gene, in the binary vector.

Synthetic sequences (such as from MAB14, nucleotide SEQ ID NO: 23, encoding the protein SEQ ID NO: 219) of some of the cloned polynucleotides were requested from a commercial supplier (GeneArt, GmbH). To optimize the coding sequence, the Tables of use of calculated codons of plant transcripts were used [examples of such Tables can be found in the Codon Use Database available online in the Hypertext Transfer Protocol: // World Wide Web (.) Kazusa (.) Or (.) Jp / codon /]. Optimized coding sequences were designed without introducing changes in the encoded amino acid sequence although using preferred codons for expression in dicotyledonous plants, mainly tomato and Arabidopsis; and in monocot plants such as corn. Such optimized sequences promote a better translation rate and therefore higher protein expression levels. Parts of the sequences were requested as the original sequences. For the optimized / non-optimized sequences additional exclusive restriction enzyme sites flanking were added to facilitate cloning genes in binary vectors.

Promoters used: Arabidopsis At6669 promoter (SEQ ID NO: 1652; which is SEQ ID NO: 61 of WO04081173 of Evogene Ltd.).

The cloned cDNA sequences are provided in SEQ ID Nos: 1530-1534, 1536-1545, 1547-1566, 1654, 1665, 1666, 1667 and 1668. The protein translation of the amplified cDNA sequence coincided exactly with the of the initial bioinformatic prediction of protein sequences. The predicted polypeptide sequences of the cloned polynucleotides are provided in SEQ ID Nos: 201, 212, 284, 213, 217, 317, 219, 221, 224, 225, 226, 227, 229, 237, 203, 247, 252, 205, 265, 267, 271, 277, 207, 208, 211, 283, 1655, 311, 334 and 254.

EXAMPLE 4

TRANSFORMATION OF AGROBACTERIUM TUMEFACIENS CELLS WITH BINARY VECTORS CARRYING ASSUMED GENES OF TEAB

Each of the binary vectors described in Example 3 above was used for the transformation of Agrobacterium cells. Two additional binary constructs, which had a GUS / Luciferase reporter gene that replaced the TEAB gene (located downstream of the At6669 promoter), were used as negative controls.

Binary vectors were introduced into competent Agrobacterium tumefaciens GV301 or LB4404 cells (approximately 109 cells / ml) by electroporation. Electroporation was performed using a MicroPulser electroporator (Biorad), 0.2 cm cuvettes (Biorad) and the EC-2 electroporation program (Biorad). The treated cells were cultured in LB liquid medium at 28 ° C for 3 hours, and then plated on LB agar supplemented with gentamicin (50 mg / l; for strains of Agrobacterium GV301) or streptomycin (300 mg / l; for strain of Agrobacterium LB4404) and kanamycin (50 mg / l) at 29 ° C for 48 hours. Agrobacterium colonies that developed on the selective media were analyzed by PCR using the primers described above (Example 3) with respect to the identification of positive colonies of binary vectors. The products resulting from the PCR were isolated and sequenced as described in Example 3 above, to verify that the correct TEAB sequences were properly introduced into Agrobacterium cells.

EXAMPLE 5

TRANSFORMATION OF THALIAN ARABIDOPSIS PLANTS WITH TEABT GENES ASSUMPTIONS

Arabidopsis thaliana Columbia plants (To plants) were transformed using the Floral Immersion procedure described by Clough and Bent (10) and by Desfeux et al. (11), with minor modifications. In summary, T0 Plants were planted in 250 ml pots loaded with a wet peat based growth mixture. The pots were covered with aluminum foil and with a plastic dome, kept at 4 ° C for 3-4 days, then uncapped and incubated in a culture chamber at 18-24 ° C with light / dark cycles of 16 /8 hours. The T0 plants were ready for transformation six days before the anthesis.

Simple colonies of Agrobacterium were generated that carried the binary constructs, such as those generated in Example 4 above. Colonies were grown in LB medium supplemented with kanamycin (50 mg / l) and gentamicin (50 mg / l). The cultures were incubated at 29 ° C for 48 hours with intense agitation and then centrifuged at 4000 rpm for 5 minutes. The sediments comprising Agrobacterium cells were resuspended in a transformation medium containing Murashige-Skoog (2.15 g / l) diluted to 50% (Duchefa); 0.044 µM benzylamino purine (Sigma); Vitamins Gambourg B5 112 /g / L (Sigma); 5% sucrose; and Silwet L-77 0.2 ml / l (OSI Specialists, CT) in doubly distilled water, at a pH of 5.7.

The transformation of To plants was carried out by inverting each plant in an Agrobacterium suspension, so that the plant tissue above the ground was submerged for 3-5 seconds. Each inoculated To plant was immediately placed in a plastic tray, then covered with a transparent plastic dome to maintain moisture and kept in the dark at room temperature for 18 hours, to facilitate infection and transformation. Then, the transformed (transgenic) plants were uncovered and transferred to a greenhouse for recovery and maturation. Transgenic To plants were grown in the greenhouse for 3-5 weeks until the silicones turned brown and dried. The seeds were collected from the plants and kept at room temperature until planting.

To generate T1 and T2 transgenic plants that carried the genes, the surface of the seeds collected from the transgenic To plants was sterilized by immersing them in 70% ethanol for 1 minute, followed by immersion in 5% sodium hypochloride and 0 triton, 05% for 5 minutes. The seeds with the sterilized surface were carefully washed in sterile distilled water and then placed in culture plates containing 50% diluted Murashige-Skoog (Duchefa); 2% sucrose; 0.8% vegetable agar; 50 mM kanamycin; and 200 mM carbenicillin (Duchefa). The culture plates were incubated at 4 ° C for 48 hours, then transferred to a culture room at 25 ° C for another week of incubation. Vital Arabidopsis T1 plants are transferred to recent culture plates for another week of incubation. After incubation, T1 plants were removed from the culture plates and planted in 250 ml pots containing growth mixture. The transgenic plants were allowed to develop in a greenhouse until maturity. Seeds collected from T1 plants were grown and grown to maturity as T2 plants under the same conditions as those used for the cultivation and growth of T1 plants.

EXAMPLE 6

TEAB IMPROVED IN TISSUE CULTURE TEST

Test 1: Plant growth with Osmotic stress (PEG) under tissue culture conditions - Osmotic stress conditions (PEG) resemble those of high osmolarity found during drought (for example, 25% PEG8000). One of the consequences of drought is the induction of osmotic stress in the area surrounding the roots; therefore, in many scientific studies, PEG serves to simulate drought.

Seeds were seeded with the surface sterilized in base medium (50% Murasige-Skoog (MS) medium supplemented with 0.8% vegetable agar as solidifying agent) in the presence of Kanamycin (to select only transgenic plants). the plates were transferred for 2-3 days at 4 ° C for stratification and then grown at 25 ° C with daily cycles of 12 hours of light, 12 hours of darkness, for 7 to 10 days, at this time, randomly selected seedlings they were carefully transferred to hot plates with 25% PEG in MS 0.5 medium or in normal conditions (MS 0.5 medium) Each plate contained 5 seedlings of the same event, and 3-4 different plates (copies) for each event For each polynucleotide of the invention, at least four transformation events independent of each construction were analyzed Plants that expressed the polynucleotides of the invention were compared with the average measurement of the plants of Control: Simulated transgenic plants expressing the uidA indicator gene (GUS Intron - GUI) under the same promoter were used as control.

Digital imaging - A laboratory imaging system was used, consisting of a digital SLR camera (Canon EOS 300D) connected to a 55mm focal length lens (Canon EF-S series), mounted on a device player (Kaiser RS), which included 4 light units (4x 150 watt light bulbs) and located in a dark room, to capture images of seedlings grown on square plates with agar.

The image capture process was repeated every 7 days starting from day 0 to day 14. The same camera connected to a 24mm focal length lens (Canon EF series), placed in a custom-made iron mount was used To take pictures

An image analysis system was used, consisting of a personal desktop computer (Intel P4 3.0 GHz processor) and a public domain program - ImageJ 1.37 (Java-based image processing program that was developed in the US National Institutes of Health and free on the Internet in the Hypertext Transfer Protocol: // rsbweb (.) Nih (.) Gov /). The images were captured in 6 Megapixel resolution (3072 x 2048 pixels) and stored in a low compression JPEG (Joint Photographic Experts Group standard) format. Then, the analyzed data were saved in text files and processed using the JMP statistical software (SAS Institute).

Seedling analysis - Using the digital analysis, seedling data were calculated, including leaf area, root coverage and root length.

The Relative Growth Rate (TCR) was calculated according to the following formula I.

Formula I:

Relative growth area rate = ( Area / (t) * (1 / Area t0)

t is the current day of the analyzed image subtracted from the initial day (t-t0). Therefore, the relative growth area rate is in units of 1 / day and the longitudinal growth rate is in units of 1 / day.

At the end of the experiment, the seedlings were removed from the media and weighed to determine the fresh weight of the plant. The Relative Growth Rate was determined by comparing the leaf area, root length and root coverage between each pair of sequential photographs, and the results were used to resolve the effect of the introduced gene on plant vigor, under conditions of osmotic stress. , as well as in optimal conditions. Similarly, the effect of the introduced gene on biomass accumulation, under stress conditions, as well as in optimal conditions, was determined by comparing the fresh weight of the plants with the control plants (GUI).

Statistical analyzes - To identify genes and higher constructs, the results of independent transformation events were evaluated for the overall influence of the gene (effect of the gene) and for each of the events tested (best event). Student's t-test was applied, using the significance of p <0.05 or p <0.1. The JMP statistical software package (Version 5.2.1, SAS Institute Inc., Cary, NC, USA) was used.

Experimental Results

The polynucleotide sequences of the invention were tested for various desired traits.

Tables 5-6 represent analysis of the Foliar Area in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669 under conditions of 25% PEG. Each Table represents an independent experiment, using 4 independent events per gene. Unrelated genes by the same letter as that of the control (A, B) are significantly different from those of the control, indicating A a difference at a level of significance of P <0.05 and A * a difference at a level of significance of P <0.1.

Tables 7-9 represent analysis of the Radical Coverage in plants that overexpress the polynucleotides of the invention under the regulation of the 6669 promoter under 25% PEG conditions. Each Table represents an independent experiment, using 4 independent events per gene. Unrelated genes by the same letter as that of the control (A, B) are significantly different from those of the control.

Tables 10-11 represent Radical Length analysis in plants that overexpress the polynucleotides of the invention under the regulation of the 6669 promoter under 25% PEG conditions. Each Table represents an independent experiment, using 4 independent events per gene. Unrelated genes by the same letter as that of the control (A, B) are significantly different from those of the control.

Tables 12-13 represent TCR (Relative Growth Rate) analysis of the Foliar Area in plants that overexpress the polynucleotides of the invention under the regulation of the 6669 promoter under 25% PEG conditions. Each Table represents an independent experiment, using 4 independent events per gene. Unrelated genes by the same letter as that of the control (A, B) are significantly different from those of the control.

Tables 14-18 represent TCR analysis of root coverage in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669 under conditions of 25% PEG. Each Table represents an independent experiment, using 4 independent events per gene. Unrelated genes by the same letter as that of the control (A, B) are significantly different from those of the control

Tables 19-21 represent TCR Radical Length analysis in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669 with 25% PEG. Each Table represents an independent experiment, using 4 independent events per gene. Unrelated genes by the same letter as that of the control (A, B) are significantly different from those of the control.

Tables 22-23 represent analysis of the Fresh Weight of Plants in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669 with 25% PEG. Each Table represents an independent experiment, using 4 independent events per gene. Unrelated genes by the same letter as that of the control (A, B) are significantly different from those of the control.

Table 22

Gen Id

Fresh Plant Weight [g], 25% PEG

LSM

Significance Best LSM event Significance % improvement of the best event

GUI

0.20 B 0.20 B

MAB15

 0.25 B 0.30 TO 51

MAB18

 0.21 B 0.26 TO 33

Table 22; LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 23

Gen Id

Fresh Plant Weight [g], 25% PEG

LSM

Significance Best LSM event Significance % improvement of the best event

GUI

0.18 B 0.18 B

MAB17

 0.22 B 0.29 TO 66

MAB3_GA

 0.18 B 0.27 TO 53

Table 23; LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

10 Tables 24-27 represent analyzes of the Foliar Area Plants in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669 under normal conditions. Each Table represents an independent experiment, using 4 independent events per gene. Unrelated genes by the same letter as that of the control (A, B) are significantly different from those of the control.

Tables 28-31 represent analysis of Radical Coverage in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669 under normal conditions. Each Table represents an independent experiment, using 4 independent events per gene. Unrelated genes by the same letter as that of the control (A, B) are significantly different from those of the control.

Table 29

Radical Coverage [cm2], Normal conditions

Gen Id

Day 7 from the plantation

LSM

Significance Best LSM event Significance % improvement of the best event

GUI

5.40 B 5.40 B

MAB100

 5.05 B 7.06 TO 31

Table 29: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Tables 32-33 represent Radical Length analysis in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669 under normal conditions. Each Table represents an independent experiment, using 4 independent events per gene. Unrelated genes by the same letter as that of the control (A, B) are significantly different from those of the control.

Tables 34-36 represent TCR analysis of the Foliar Area in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669 under normal conditions. Each Table represents an independent experiment, using 4 independent events per gene. Unrelated genes by the same letter as that of the control (A, B) are significantly different from those of the control.

Tables 37-41 represent TCR analysis of Radical Coverage in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669 under normal conditions. Each Table represents an independent experiment, using 4 independent events per gene. Unrelated genes by the same letter as that of the control (A, B) are significantly different from those of the control.

Tables 42-46 represent analysis of the Radical Length TCR in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669 under normal conditions. Each Table represents an independent experiment, using 4 independent events per gene. Unrelated genes by the same letter as that of the control (A, B) are significantly different from those of the control.

Table 42

Radical Length TCR [cm / day], Normal conditions

Gen ID

Day 7 from the plantation

LSM

Significance* Best LSM event Significance* % improvement of the best event

GUI

1.07 B 1.07 B

MAB10

 1.29 B 2.01 TO 88

Table 42: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 43

Radical Length TCR [cm / day], Normal conditions

Gen ID

Day 7 from the plantation

LSM

Significance* Best LSM event Significance* % improvement of the best event

GUI

0.17 B 0.17  B

MAB1

0.26 TO 0.34  TO 93

MAB15

0.32 TO 0.45  TO 156

MAB17

0.24 TO 0.28  TO 61

MAB18

0.30 TO 0.41  TO 136

MAB146

0.26 TO 0.34  TO 93

Table 43: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 45

Radical Length TCR [cm / day], Normal conditions

Gen ID

Day 14 from the plantation

LSM

Significance* Best LSM event Significance* % improvement of the best event

GUI

0.11 B 0.11  B

MAB32

0.11 B 0.15  TO 35

MAB35

0.11 B 0.20  TO 76

MAB4

0.11 B 0.17  TO fifty

MAB146

0.15 TO 0.19  TO 71

Table 45: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Tables 47-48 represent Fresh Plant Weight analysis in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669 under normal conditions. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B) are significantly different from those of the control.

Table 47

Fresh Plant Weight [g], Normal conditions

Gen ID

Day 14 from the plantation

LSM

Significance* Best LSM event Significance* % improvement of the best event

GUI

0.15 B 0.15 B

MAB15

 0.24 TO 0.28 TO 93

MAB17

 0.21 TO 0.25 TO 73

MAB18

 0.22 TO 0.29 TO 101

Table 47: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 48

Fresh Plant Weight [g], Normal conditions

Gen ID

Day 14 from the plantation

LSM

Significance* Best LSM event Significance* % improvement of the best event

GUI

0.20 B 0.20  B

MAB100

 0.28 TO* 0.33 TO 62

MAB134

0.23 B 0.34  TO 64

MAB13

0.31 TO 0.35  TO 73

MAB15

0.38 TO 0.42  TO 106

MAB17

0.37 TO 0.53  TO 159

MAB3_GA

 0.28 TO* 0.40 TO 94

Table 48: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

10 Test 2: Growth of Nitrogen deficit plants, under tissue culture conditions - The authors of the present invention have discovered that the EUN (Nitrogen Utilization Efficiency) test is relevant for the evaluation of candidate TEAB genes , since a deficit of the EUN promotes root lengthening, increases root coverage and allows to detect the potential of the plant to generate a better root system in drought conditions. In addition, in the literature (Wesley et al., 2002 Journal of Experiment Botany Vol. 53, No.

15 366, p. 13-25) there are indications that the biological mechanisms of EUN and drought tolerance are related.

Sterilized seeds on the surface were sown in basal medium [50% Murashige-Skoog Medium (MS) supplemented with 0.8% vegetable agar as solidifying agent] in the presence of kanamycin (to select only 20 transgenic plants). After sowing, the plates were transferred for 2-3 days at 4 ° C for stratification and then grown at 25 ° C with daily cycles of 12 hours of light, 12 hours of darkness, for 7 to 10 days. At this time, the randomly selected seedlings were carefully transferred to plates containing nitrogen limiting conditions: MS 0.5 medium in which the combined nitrogen concentration (NH4NO3 and KNO3) is 0.75 mM (conditions with deficit of nitrogen) or to plates carrying normal nitrogen conditions: 0.5 MS medium in which the combined nitrogen concentration (NH4NO3 and KNO3) is 3 mM (normal nitrogen concentration). All tissue culture experiments were developed at the same time (EUN, PEG and Normal). The results of growth under normal conditions for EUN are the same as for PEG and are presented in test 1. Each plate contains 5 seedlings of the same event, and 3-4 different plates (copies) for each event. For each polynucleotide of the invention at least four events were analyzed.

30 independent transformation of each construction. The plants expressing the polynucleotides of the invention are compared with the average measurement of the control plates (GUI - which carries the GUS gene under the same promoter) used in the same experiment.

Digital imaging and statistical analysis - Parameters were measured and analyzed as described in Test 1 above.

Experimental results - The polynucleotide sequences of the invention were tested for various desired traits.

Tables 49-53 represent analyzes of the Foliar Area in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669 under conditions with nitrogen deficit. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B) are significantly different from those of the control.

Table 51

Foliar Area [cm2], EUN 0.75 mM

Gen ID

Day 7 from the plantation

LSM

Significance* Best LSM event Significance* % improvement of the best event

GUI

0.34 B 0.34 B

MAB17

 0.32 B 0.44 TO 31

MAB3_GA

 0.32 B 0.44 TO 31

Table 51: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Tables 54-57 represent analysis of Radical Coverage in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669 under nitrogen deficit conditions. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B) are significantly different from those of the control.

Tables 58-61 represent analysis of the Radicular Length in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669 under conditions with nitrogen deficit. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B) are significantly different from those of the control.

Table 58

Radicular Length [cm], EUN 0.75 mM

Gen ID

Day 7 from the plantation

LSM

Significance* Best LSM event Significance* % improvement of the best event

GUI

6.31 B 6.31 B

MAB44

 5.34 B 7.07 TO 12

Table 58: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 61

Radicular Length [cm], EUN 0.75 mM

Gen ID

Day 7 from the plantation

LSM

Significance* Best LSM event Significance* % improvement of the best event

GUI

4.87 B 4.87 B

MAB66

 5.27 B 5.74 TO 18

Table 61: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Tables 62-64 represent TCR analysis of the Foliar Area in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669 under nitrogen deficit conditions. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B) are significantly different from those of the control.

Table 63

Foliar Area TCR [cm / day], EUN 0.75 mM

Gen ID

Day 7 from the plantation

LSM

Significance* Best LSM event Significance* % improvement of the best event

GUI

0.80 B 0.80  B

MAB18

0.87 B 1.24  TO 56

MAB32

0.94 B 1.53  TO 91

MAB35

0.96 B 1.21  TO 51

MAB4

0.71 B 0.81  B one

MAB46

0.64 B 0.75  B -7

MAB146

0.82 B 1.04  B 30

Table 63: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Tables 65-69 represent TCR analysis of Radical Coverage in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669 under conditions with nitrogen deficit. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B, C) are significantly different from those of the control.

Table 67

TCR Radical Coverage [cm / day], EUN 0.75 mM

Gen ID

Day 7 from the plantation

LSM

Significance* Best LSM event Significance* % improvement of the best event

GUI

2.30 B 2.30  B

MAB100

2.85 B 4.02  TO 74

MAB134

4.27 TO 5.99  TO 160

MAB13

3.95 TO 4.84  TO 110

MAB15

 3.05 TO* 3.97 TO 73

MAB17

2.96 B 3.76  TO 63

Table 67: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 69

TCR Radical Coverage [cm / day], EUN 0.75 mM

Gen ID

Day 7 from the plantation

LSM

Significance* Best LSM event Significance* % improvement of the best event

GUI

1.60 B 1.60 B

MAB137

 2.19 TO 2.55 B 60

MAB43

 2.00 B 2.75 TO 72

MAB50

 2.26 TO 3.28 TO 105

MAB6

2.45 TO 2.96 TO 85

MAB66

 1.81 B 2.87 TO 80

MAB99

 2.25 TO 3.73 TO 133

Table 69: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Tables 70-74 represent analysis of the Radical Length TCR in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669 under nitrogen deficit conditions. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B) are significantly different from those of the control.

Tables 75-76 represent analysis of the Fresh Weight of the Plant in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669 under conditions with nitrogen deficit. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B), are significantly different from those of the control.

Table 75

Fresh Plant Weight [g], EUN 0.75 mM

Gen ID

Day 14 from the plantation

LSM

Significance* Best LSM event Significance* % improvement of the best event

GUI

0.15 B

MAB1

0.25 TO 0.46 TO 208

MAB6

0.20 B 0.29 TO 95

Table 75: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 76

Fresh Plant Weight [g], EUN 0.75 mM

Gen ID

Day 10 from the plantation

LSM

Significance* Best LSM event Significance* % improvement of the best event

GUI

0.15 B

MAB137

0.18 TO 0.19  TO 31

MAB50

0.16 B 0.22  TO 49

MAB6

0.16 B 0.22  TO 52

MAB66

0.15 B 0.19  TO 32

Table 76: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

10 EXAMPLE 7

TEAB IMPROVED IN GREENHOUSE TESTS

EAB Tolerance: Plant yield and growth rate at high salinity concentration in

15 greenhouse conditions - This test is based on the growth of the rosette area of plants grown in the greenhouse, as well as the yield of seeds with high salinity irrigation. The seeds were sown in medium with agar supplemented only with a selection agent (Kanamycin) and Hoagland solution under nursery conditions. Then, the T2 transgenic seedlings were transplanted in 1.7 liter trays loaded with peat and perlite. The trays were watered with running water (provided from the base of the pots). The

20 half of the plants were watered with a saline solution (40-80 mM NaCl and 5 mM CaCl2) to induce salinity stress (stress conditions). The other half of the plants continued to be irrigated with running water (normal conditions). All plants grew in the greenhouse until the plants reached the mature seed phase, then they were collected (the tissue above the ground) and weighed (immediately or after drying in an oven at 50 ° C for 24 hours). High salinity conditions were obtained by watering with a solution

25 containing 40-80 mM NaCl (growth conditions with "EAB") and then purchased with regular growth conditions.

The size, growth rate, seed yield and weight of 1,000 seeds, dry matter and the harvest index (IC-seed yield / dry matter) of the plants were analyzed. The performance of

30 transgenic plants were compared with that of control plants that grew in parallel under the same conditions. As a control, simulated transgenic plants that expressed the uidA indicator gene (GUS Intron-GUI) under the same promoter were used.

The experiment was planned in a nested randomized plot distribution. The high salinity conditions were achieved by watering with a solution containing 40-80 mM NaCl (growth conditions with "EAB").

Digital imaging - A laboratory imaging system was used, which consisted of a digital SLR camera (Canon EOS 300D) coupled to a 55mm focal length lens (Canon EF-S series), mounted on a device player (Kaiser RS), which included 4 light units (4x 150-watt light bulbs) to capture images of seedlings.

The image capture process was repeated every 2-3 days starting on day 1 after sowing until day

10. The same camera coupled with a 24mm focal length lens (Canon EF series), was placed in a custom-made iron mount, used to capture images of larger plants that were grown in white planters in a greenhouse with controlled environment (as seen in Figures 2a-b). The planters, which were square, included 1.7 liter trays. During the capture process, the trays were placed under the iron assembly, avoiding direct sunlight and shadow casting. This process was repeated every 2-3 days for up to 10 days.

An image analysis system was used, consisting of a personal desktop computer (Intel P4 3.0 GHz processor) and a public domain program - ImageJ 1.37 (Java-based image processing program that was developed in the US National Institutes of Health and free on the Internet in the Hypertext Transfer Protocol: // rsbweb (.) Nih (.) Gov /). The images were captured in 6 Megapixel resolution (3072 x 2048 pixels) and stored in a low compression JPEG (Joint Photographic Experts Group) format. Then, the analyzed data were saved in text files and processed using the JMP statistical software (SAS Institute).

Analysis of the vegetative parameters - Using the digital analyzes the data of the leaves were calculated, including the average leaf area, the diameter of the rosette and the area of the rosette. The Relative Growth Rate (TCR) of the rosette parameters was calculated according to Formula I, as described in Example 6. On day 80 after planting, the plants were collected and allowed to dry at 30 ° C in A drying chamber The biomass and the weight of the seeds of each plot were separated, measured and divided by the number of plants. The dry weight is = the total weight of the vegetative part above the ground (excluding roots) after drying at 30 ° C in a drying chamber; Seed Yield per plant is = to the weight of total seed per plant (g).

The weight of 1000 seeds was determined as follows: the seeds were disseminated in a glass tray and a photo was taken. Each sample was weighed and then, using the digital analysis, the number of seeds of each sample was calculated. The weight of 1000 seeds was calculated using formula II:

Formula II

Weight of 1000 Seeds = number of seeds in the sample / weight sample X 1000

Harvest Index - The harvest index was calculated using Formula III

Formula III:

Harvest Index = Average seed yield per plant / average dry weight

Each construction was validated in its T2 generation. As a control, transgenic plants expressing the uidA indicator gene (GUI) under the same promoter were used.

Statistical analyzers - To identify genes that confer significantly improved tolerance to abiotic stress or an elongated root architecture, the results obtained from the transgenic plants were compared with those obtained from the control plants. To identify genes and constructs with superior behavior, the results of the independent transformation events tested were analyzed separately. In addition, genes and constructions were also analyzed taking into account the results obtained in all independent transformation events tested in the specific construction For the analysis of the genes against the controls, the Student t test was applied, using a significance of P  0.05 or P <0.1. The JMP statistical software package (Version 5.2.1, SAS Institute Inc., Cary, NC, USA) was used.

Experimental Results

The polynucleotide sequences of the invention were tested for various desired traits.

Tables 77-86 represent analyzes of the Rosette Area in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B), are significantly different from those of the control.

Table 77

Gen ID

Rosette Area [cm2] 80 mM NaCl, Day 3 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.58 B 0.58  B

MAB20

0.59 B 0.84  TO 43

MAB50

0.57 B 0.88  TO 51

Table 77: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 78

Gen ID

Rosette Area [cm2] 80 mM NaCl, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

1.27 B 1.27  B

MAB20

 1.20 B 1.73 to 36

MAB50

 1.21 B 2.04 to 61

Table 78: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 79

Gen ID

Rosette Area [cm2] 80 mM NaCl, Day 8 from planting

 LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

3.62 B 3.62  B

MAB20

3.97 B 5.18  TO 43

MAB50

3.88 B 6.11  TO 69

Table 79: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 80

Gen ID

Rosette Area [cm2] 80 mM NaCl, Day 10 from planting

 LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

7.22 B 7.22  B

MAB50

 6.75 B 10.18 TO 41

Table 80: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 81 Table 82

Gen ID

Rosette Area [cm2] 80 mM NaCl, Day 3 from planting

 LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

1.63 B 1.63  B

MAB1

2.03 TO 2.29  TO 40

MAB6

1.34 B 2.40  TO 47

Table 81: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Gen ID

Rosette Area [cm2] 80 mM NaCl, Day 5 from planting

 LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

2.88 B 2.88  B

MAB1

3.41 TO* 3.76 TO 31

Table 82: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 83

Gen ID

Rosette Area [cm2] 80 mM NaCl, Day 3 from planting

 LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.73 B 0.73  B

MAB1

0.77 B 0.91  TO 25

Table 83: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 84

Gen ID

Rosette Area [cm2] 80 mM NaCl, Day 5 from planting

 LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

1.41 B 1.41  B

MAB1

1.62 TO* 2.02 TO 44

NAB17

1.14 B 1.80  TO 28

Table 84: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 85 Table 86

Gen ID

Rosette Area [cm2] 80 mM NaCl, Day 8 from planting

 LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

2.37 B 2.37  B

MAB1

2.59 B 3.56  TO fifty

MAB13

2.45 B 3.44  TO Four. Five

MAB17

 1.96 C 3.10 TO 31

Table 85: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Gen ID

Rosette Area [cm2] 80 mM NaCl, Day 10 from planting

 LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

4.67 B 4.67  B

MAB1

5.37 TO* 7.93 TO 70

MAB15

4.78 B 6.08  TO 30

MAB17

4.02 B 6.19  TO 32

MAB3_GA

4.39 B 6.07  TO 30

Table 86: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Tables 87-96 represent analysis of the Rosette Diameter in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B), are significantly different from those of the control.

Table 87

Gen ID

Rosette Diameter [cm] 80 mM NaCl, Day 3 from planting

 LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

1.50 B 1.50  B

MAB50

1.35 B 1.80  TO twenty

Table 87: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

10 Table 88

Gen ID

Rosette Diameter [cm] 80 mM NaCl, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

2.05 B 2.05  B

MAB50

 1.82 C 2.44 TO 19

Table 88: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 89 Table 90

Gen ID

Rosette Diameter [cm] 80 mM NaCl, Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

3.23 B 3.23  B

MAB50

3.16 B 4.12  TO 27

Table 89: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Gen ID

Rosette Diameter [cm] 80 mM NaCl, Day 10 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

4.47 B 4.47  B

MAB50

4.20 B 5.31  TO 19

Table 90: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 91

Gen ID

Rosette Diameter [cm] 80 mM NaCl, Day 3 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

2.25 B 2.25 B

MAB1

2.60 TO 2.78 TO 2. 3

Table 91: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 92

Gen ID

Rosette Diameter [cm] 80 mM NaCl, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

2.87 B 2.87  B

MAB1

3.27 TO* 9.25 TO 223

MAB20

2.63 B 9.69  TO 238

MAB6

2.51 B 10.00 TO 249

Table 92: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 93

Gen ID

Rosette Diameter [cm] 80 mM NaCl, Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

4.90 B 4.90 B

MAB6

4.35 B 6.26 TO 28

Table 93: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 94 Table 95

Gen ID

Rosette Diameter [cm] 80 mM NaCl, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

2.05 B 2.05 B

MAB1

2.22 B 2.55 TO 25

Table 94: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Gen ID

Rosette Diameter [cm] 80 mM NaCl, Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

2.56 B 2.56 B

MAB1

2.78 B 3.29 TO 29

MAB3 GA

2.56 B 3.04 TO 19

Table 95: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 96

Gen ID

Rosette Diameter [cm] 80 mM NaCl, Day 10 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

3.52 B 3.52  B

MAB1

3.79 B 4.76  TO 35

MAB17

3.24 B 4.14  TO 17

MAB3_GA

3.44 B 4.12  TO 17

Table 96: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

5 Tables 97-105 represent analyzes of the Foliar Average Area in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B), are significantly different from those of the control.

10 Table 97

Gen ID

Average Foliar Area [cm2] 80 mM NaCl Day 3 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.10 B 0.10  B

MAB25

0.10 B 0.13  TO 30

Table 97: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 98 Table 99

Gen ID

Average Foliar Area [cm2] 80 mM NaCl Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.16 B 0.16  B

MAB50

0.15 B 0.23  TO Four. Five

Table 98: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Gen ID

Average Foliar Area [cm2] 80 mM NaCl Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.45 B 0.45  B

MAB50

0.41 B 0.61  TO 3. 4

Table 99: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 100

Gen ID

Average Foliar Area [cm2] 80 mM NaCl Day 10 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.74 B 0.74  B

MAB50

0.66 B 0.92  TO 25

Table 100: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 101

Gen ID

Average Foliar Area [cm2] 80 mM NaCl Day 3 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.20 B 0.20 B

MAB1

0.25 TO 0.28 TO 43

MAB6

0.18 B 0.30 TO 51

MAB7

0.23 B 0.27 TO 36

Table 101: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 102

Gen ID

Average Foliar Area [cm2] 80 mM NaCl Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.69 B 0.69  B

MAB1

0.80 TO* 0.86  TO* 24

MAB20

0.62 B 0.87  TO 25

MAB6

0.59 B 0.99  TO 44

Table 102: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 103 Table 104

Gen ID

Average Foliar Area [cm2] 80 mM NaCl Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.20 B 0.20  B

MAB1

0.22 B 0.27  TO 30

MAB17

 - - 0.25 TO twenty-one

Table 103: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Gen ID

Average Foliar Area [cm2] 80 mM NaCl Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.28 B 0.28  B

MAB1

0.30 B 0.37  TO 33

MAB17

0.24 B 0.34  TO 22

Table 104: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 105

Gen ID

Average Foliar Area [cm2] 80 mM NaCl Day 10 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.49 B 0.49  B

MAB1

0.55 B 0.76  TO 53

MAB15

0.52 B 0.63  TO 26

MAB17

0.45 B 0.64  TO 28

Table 105: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

5 Tables 106-111 represent TCR analysis of the Rosette Area [cm2] of plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B), are significantly different from those of the control.

10 Table 106

Gen ID

TCR of the Rosette Area [cm2] 80 mM NaCl, Day 3 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.73 B 0.73  B

MAB10

1.21 B 1.86  TO 156

MAB14

1.31 B 1.80  TO 149

MAB2

1.59 TO 2.24  TO 208

MAB20

1.87 TO 2.33  TO 221

MAB25

 1.44 TO 1.63 TO* 125

MAB36

1.49 TO 1.89  TO 161

MAB43

1.73 TO 3.85  TO 430

MAB44

1.76 TO 2.51  TO 246

MAB50

1.37 TO* 1.57  TO* 117

MAB9

1.47 TO 1.75  TO 141

Table 106: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 107

Gen ID

TCR of the Rosette Area [cm2] 80 mM NaCl, Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.61 B 0.61  B

MAB10

 0.75 TO* 0.91 TO fifty

MAB14

0.79 TO 0.86  B 42

MAB19

0.78 TO 0.85  TO 41

MAB2

0.80 TO 0.93  TO 54

MAB20

0.79 TO 0.98  TO 61

MAB36

0.83 TO 0.95  TO 56

MAB44

 0.75 TO* 0.84 TO 38

MAB50

 0.76 TO* 0.83 B 38

MAB6

0.82 TO 0.99  TO 64

MAB7

0.78 TO 0.87  TO 44

MAB9

0.77 TO 0.84  TO 38

Table 107: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 108

Gen ID

TCR of the Rosette Area [cm2] 80 mM NaCl, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.38 B 0.38 B

MAB6

0.37 B 0.51 TO 33

Table 108: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 109

Gen ID

TCR of the Rosette Area [cm2] 80 mM NaCl, Day 3 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.88 B 0.88  B

MAB18

 0.99 TO* 1.24 TO 41

Table 109: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 110 Table 111

Gen ID

TCR of the Rosette Area [cm2] 80 mM NaCl, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.47 B 0.47  B

MAB1

0.55 TO 0.64  TO 38

MAB13

 0.52 TO 0.54 TO* 16

MAB17

 0.52 TO 0.54 TO* 17

MAB18

0.53 TO 0.58  TO 24

MAB3_GA

0.53 TO 0.62  TO 33

MAB32

0.52 TO* 0.54  TO* 17

MAB35

0.54 TO 0.57  TO 22

MAB4

0.51 TO* 0.51  TO* 10

MAB46

 0.52 TO* 0.55 TO 19

MAB146

0.54 TO 0.55  TO 19

MAB99

0.53 TO 0.57  TO 2. 3

Table 110: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Gen ID

TCR of the Rosette Area [cm2] 80 mM NaCl, Day 3 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.49 B 0.49  B

MAB1

0.53 B 0.62  TO 27

MAB35

0.57 TO* 0.59  TO* 22

MAB46

0.55 B 0.63  TO 30

Table 111: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Tables 112-118 represent analysis of the Rosette Diameter TCR in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B), are significantly different from those of the control.

Table 112

Gen ID

Rosette Diameter TCR [cm] 80 mM NaCl, Day 3 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.28 B

MAB2

0.41 B 0.80  TO 184

MAB43

0.46 B 0.83  TO 195

MAB44

0.40 B 0.73  TO 160

Table 112: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 113

Gen ID

Rosette Diameter TCR [cm] 80 mM NaCl, Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.19 B 0.19  B

MAB1

0.22 B 0.24  B 25

MAB10

0.25 TO 0.29  TO 49

MAB14

0.23 TO 0.25  TO 31

MAB19

0.24 TO 0.26  TO 37

MAB2

0.24 TO 0.26  TO 3. 4

MAB20

0.25 TO 0.29  TO 52

MAB25

0.24 TO 0.27  TO 42

MAB36

0.25 TO 0.28  TO Four. Five

MAB43

0.22 B 0.25  B 28

MAB50

0.25 TO 0.28  TO 46

MAB6

0.24 TO 0.27  TO 41

MAB7

0.22 B 0.27  TO 38

MAB9

0.23 TO 0.26  TO 3. 4

Table 113: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 114 Table 115

Gen ID

Rosette Diameter TCR [cm] 80 mM NaCl, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.14 B 0.14  B

MAB10

0.14 B 0.31  TO 122

MAB20

0.13 B 0.21  TO 49

MAB25

0.15 B 0.33  TO 138

MAB9

0.15 B 0.20  TO Four. Five

Table 114: LSM = Mean least squares; % improvement = compared to the control (GUI); A means

Gen ID

Rosette Diameter TCR [cm] 80 mM NaCl, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Gen ID

Rosette Diameter TCR [cm] 80 mM NaCl, Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.21 B 0.21  B

MAB20

0.23 B 0.34  TO 67

MAB9

0.22 B 0.44  TO 114

Table 115: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 116

Gen ID

Rosette Diameter TCR [cm] 80 mM NaCl, Day 3 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.34 B 0.34  B

MAB18

0.37 B 0.46  TO 35

MAB3_GA

0.34 B 0.43  TO 26

MAB35

0.43 TO 0.55  TO 62

MAB46

0.39 B 0.49  TO 42

MAB99

0.34 B 0.43  TO 26

Table 116: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 117

Gen ID

Rosette Diameter TCR [cm] 80 mM NaCl, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.16 B 0.16 B

MAB1

0.22 TO 0.26 TO 66

MAB18

 0.20 TO* 0.23 TO* 44

MAB46

 0.25 TO 0.45 TO 185

MAB146

 0.20 TO* 0.22 TO* 42

Table 117: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 118

Gen ID

Rosette Diameter TCR [cm] 80 mM NaCl, Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.08 B 0.08 B

MAB35

 0.10 B 0.13 TO 57

MAB46

 0.10 B 0.14 TO 64

MAB146

 0.10 B 0.14 TO 66

MAB99

 0.10 B 0.13 TO 56

Table 118: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

10 Tables 119-121 represent TCR analysis of the Foliar Average Area [cm2] in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B), are significantly different from those of the control.

Table 119

Gen ID

TCR of the Foliar Average Area [cm2] 80 mM NaCl, Day 3 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.35 B 0.35  B

MAB14

0.34 B 0.63  TO 82

MAB25

0.44 B 0.83  TO 137

MAB36

0.43 B 0.77  TO 120

MAB6

0.24 B 0.70  TO 102

Table 119: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 120

Gen ID

TCR of the Foliar Average Area [cm2] 80 mM NaCl, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.32 B 0.32  B

MAB10

0.32 B 0.56  TO 74

Table 120: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 121

Gen ID

TCR of the Foliar Average Area [cm2] 80 mM NaCl, Day 3 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.39 B 0.39  B

MAB13

0.41 B 0.57  TO 49

MAB15

 0.46 TO* 0.54 TO 40

MAB17

0.46 TO* 0.50  TO* 30

Table 121: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

10 Table 122 represents TCR analysis of the Foliar Average Area [cm2] in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B), are significantly different from those of the control.

Table 122

Gen ID

TCR of the Foliar Average Area [cm2] 80 mM NaCl, Day 3 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.28 B 0.28  B

MAB2

0.41 B 0.80  TO 184

MAB43

0.46 B 0.83  TO 195

MAB44

0.40 B 0.73  TO 160

Table 122: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 123 represents Dry Weight Analysis (PS) per Parcel in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669. Each Table represents an independent experiment, using 20 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B), are significantly different from those of the control.

Table 123

Gen ID

Dry Weight [g] 80 mM NaCl

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

4.00 B 4.00 B

MAB1

4.92 TO 6.40 TO 60

MAB134

 4.35 B 5.35 TO 3. 4

MAB15

 4.42 B 5.57 TO 39

MAB18

 4.52 B 5.35 TO 3. 4

MAB3 GA

4.53 B 5.47 TO 37

Table 123: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Tables 124-126 represent analysis of the Weight of 1000 Seeds in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B), are significantly different from those of the control.

Table 124

Gen ID

Weight of 1000 Seeds [g] NaCl 80 mM

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.02 B 0.02  B

MAB14

0.02 B 0.03  TO 32

MAB19

0.02 B 0.03  TO 27

MAB2

0.02 B 0.03  TO 24

MAB6

0.03 TO 0.03  TO 53

Table 124: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 125

Gen ID

Weight of 1000 Seeds [g] NaCl 80 mM

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.02 B 0.02  B

MAB20

 0.02 TO* 0.02 TO 17

MAB25

0.02 B 0.02  TO twenty

MAB6

0.02 TO* 0.02 TO twenty-one

MAB7

0.02 B 0.02  TO twenty-one

MAB9

0.02 B 0.02  TO 19

Table 125: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 126

I

Weight of 1000 Seeds [g] NaCl 80 mM

LSM

% improvement of the best event LSM of the best event Significance % improvement of the best event

GUI

0.02 B 0.02 B

MAB100

 0.02 B 0.02 TO 28

MAB134

 0.02 B 0.02 TO 26

MAB17

 0.02 B 0.02 TO 2. 3

MAB18

 0.02 B 0.02 TO 17

MAB32

 0.02 B 0.02 TO 13

MAB4

0.02 B 0.02 TO 19

MAB46

 0.02 B 0.02 TO 18

MAB99

 0.02 B 0.02 TO fifteen

Table 126: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means

I

Weight of 1000 Seeds [g] NaCl 80 mM

LSM

% improvement of the best event LSM of the best event Significance % improvement of the best event

significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Tables 127-129 represent analysis of Seed Yield per Plant in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B), are significantly different from those of the control.

Table 127

Gen ID

Seed Yield per Plant [g] 80 mM NaCl

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.07 B 0.07  B

MAB44

0.11 B 0.22  TO 210

MAB50

0.11 B 0.19  TO 170

Table 127: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 128

Gen ID

Seed Yield per Plant [g] 80 mM NaCl

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.09 B 0.09 B

MAB6

0.11 TO* 0.21 TO 142

MAB9

0.09 B 0.14 TO 59

Table 128: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 129

Gen ID

Seed Yield per Plant [g] 80 mM NaCl

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.14 B 0.14 B

MAB1

0.19 TO 0.33 TO 139

MAB100

 0.17 B 0.24 TO 79

Table 129: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 130 represents analysis of the Harvest Index in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669 under conditions with nitrogen deficit. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B), are significantly different from those of the control.

Table 130

Gen ID

80 mM NaCl Harvest Index

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.11 B 0.11  B

MAB25

0.16 B 0.26  TO 139

MAB44

 0.20 TO* 0.30 TO 174

MAB7

0.12 B 0.29  TO 172

Table 130: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1.

Gen ID

80 mM NaCl Harvest Index

LSM

Significance LSM of the best event Significance % improvement of the best event

SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Tables 131-140 represent analysis of the Rosette Area in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B), are significantly different from those of the control.

Table 131

Gen ID

Rosette Area [cm2] Normal conditions, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

1.37 B 1.37 B

MAB1

1.43 B 1.80 TO 31

MAB9

1.32 B 1.74 TO 27

Table 131: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 132

Gen ID

Rosette Area [cm2] Normal conditions, Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

4.73 B 4.73 B

MAB1

4.95 B 6.45 TO 36

Table 132: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 133

Gen ID

Rosette Area [cm2] Normal conditions, Day 10 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

8.45 B 8.45 B

MAB1

8.87 B 11.11  TO 31

Table 133: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 134

Gen ID

Rosette Area [cm2] Normal conditions, Day 3 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

1.65 B 1.65  B

MAB1

2.09 TO 2.27  TO 37

MAB36

1.65 B 2.58  TO 56

MAB7

1.83 B 2.81  TO 70

Table 134: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 135

Gen ID

Rosette Area [cm2] Normal conditions, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

2.93 B 2.93  B

MAB1

3.60 TO* 3.78  TO* 29

Gen ID

Rosette Area [cm2] Normal conditions, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

MAB36

2.91 B 4.55  TO 55

MAB7

3.14 B 4.69  TO 60

Table 135: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 136

Gen ID

Rosette Area [cm2] Normal conditions, Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

7.73 B 7.73  B

MAB1

9.77 TO 10.58 TO 37

MAB36

 8.05 B 12.12 TO 57

MAB7

8.69 B 12.82 TO 66

Table 136: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 137

Gen ID

Rosette Area [cm2] Normal conditions, Day 3 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.55 B 0.55 B

MAB1

0.58 B 0.81 TO 47

MAB100

 0.60 B 0.74 TO 3. 4

MAB15

 0.65 TO* 0.90 TO 64

MAB17

 0.55 B 0.85 TO 55

Table 137: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 138

Gen ID

Rosette Area [cm2] Normal conditions, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

1.03 B 1.03 B

MAB1

1.17 B 1.54 TO 49

MAB100

 1.18 B 1.46 TO 42

MAB15

 1.23 TO 1.67 TO 62

MAB17

 1.01 B 1.59 TO 54

Table 138: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 139 Table 140

Gen ID

Rosette Area [cm2] Normal conditions, Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

2.09 B 2.09 B

MAB1

2.46 B 3.43 TO 64

MAB100

 2.29 B 2.81 TO 3. 4

MAB15

 2.60 TO 3.63 TO 73

MAB17

 2.06 B 3.35 TO 60

Table 139: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Gen ID

Rosette Area [cm2] Normal conditions, Day 10 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

4.81 B 4.81  B

MAB1

5.57 TO* 8.29 TO 72

MAB15

5.72 TO 8.05  TO 67

MAB17

4.78 B 7.50  TO 56

Table 140: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Tables 141-148 represent analysis of the Rosette Diameter in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B), are significantly different from those of the control.

Table 141

Gen ID

Rosette Diameter [cm] Normal conditions, Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

3.52 B 3.52 B

MAB1

3.58 B 4.17 TO 18

Table 141: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 142

Gen ID

Rosette Diameter [cm] Normal conditions, Day 3 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

2.28 B 2.28  B

MAB36

2.23 B 2.91  TO 28

MAB7

2.47 B 3.11  TO 36

Table 142: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 143

Gen ID

Rosette Diameter [cm] Normal conditions, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

2.99 B 2.99 B

MAB7

3.24 B 4.08 TO 36

Table 143: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 144 Table 145

Gen ID

Rosette Diameter [cm] Normal conditions, Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

5.00 B 5.00 B

MAB1

5.65 TO* 5.87 TO* 17

MAB7

5.06 B 6.32 TO 26

Table 144: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Gen ID

Rosette Diameter [cm] Normal conditions, Day 3 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

1.30 B 1.30  B

MAB15

1.48 TO 1.69  TO 30

MAB17

1.33 B 1.60  TO 2. 3

Table 146: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 146

Gen ID

Rosette Diameter [cm] Normal conditions, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

1.87 B 1.87  B

MAB1

1.86 B 2.21  TO 18

MAB15

1.96 B 2.29  TO 22

MAB17

1.78 B 2.26  TO twenty-one

Table 146: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 147

Gen ID

Rosette Diameter [cm] Normal conditions, Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

2.49 B

MAB1

2.60 B 3.14  TO 26

MAB15

2.64 B 3.17  TO 27

MAB17

2.39 B 3.09  TO 24

Table 147: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 148

Gen ID

Rosette Diameter [cm] Normal conditions, Day 10 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

3.49 B 3.49  B

MAB1

3.88 TO* 4.81 TO 38

MAB 15

3.78 B 4.52  TO 29

MAB17

3.53 B 4.45  TO 27

Table 148: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Tables 149-157 represent analysis of the Foliar Average Area in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669. Each Table represents an independent experiment, using 10 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B), are significantly different from those of the control.

Table 149 Table 150

Gen ID

Average Foliar Area [cm2] Normal conditions, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.17 B 0.17 B

MAB1

0.17 B 0.21 TO 27

Table 149: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in the

Gen ID

Average Foliar Area [cm2] Normal conditions, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

Plants are provided in Table 3 above.

Gen ID

Average Foliar Area [cm2] Normal conditions, Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.51 B 0.51 B

MAB1

0.52 B 0.69 TO 35

Table 150: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 151

Gen ID

Average Foliar Area [cm2] Normal conditions, Day 3 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.19 B 0.19  B

MAB1

0.25 TO 0.27  TO 38

MAB36

0.20 B 0.31  TO 58

MAB7

0.23 TO* 0.33 TO 67

Table 151: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 152

Gen ID

Average Foliar Area [cm2] Normal conditions, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.32 B 0.32  B

MAB1

0.38 B 0.43  TO 3. 4

MAB36

0.32 B 0.46  TO 43

MAB7

0.33 B 0.47  TO Four. Five

Table 152: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 153

Gen ID

Average Foliar Area [cm2] Normal conditions, Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.69 B 0.69  B

MAB36

0.69 B 0.93  TO 36

MAB7

0.79 B 1.17  TO 71

Table 153: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 154

Gen ID

Average Foliar Area [cm2] Normal conditions, Day 3 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.11 B 0.11  B

MAB1

0.12 B 0.15  TO 28

MAB15

0.13 B 0.17  TO 53

MAB17

0.11 B 0.15  TO 3. 4

Table 154: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1.

Gen ID

Average Foliar Area [cm2] Normal conditions, Day 3 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 155

Gen ID

Average Foliar Area [cm2] Normal conditions, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.16 B 0.16 B

MAB1

0.17 B 0.21 TO 26

MAB100

 0.18 B 0.21 TO 30

MAB15

 0.18 TO* 0.23 TO 39

MAB17

 0.16 B 0.22 TO 35

Table 155: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 156

Gen ID

Average Foliar Area [cm2] Normal conditions, Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.24 B 0.24  B

MAB1

0.28 TO* 0.37 TO fifty

MAB 15

0.29 TO* 0.37 TO 53

MAB17

0.25 B 0.34  TO 40

Table 156: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 157

Gen ID

Average Foliar Area [cm2] Normal conditions, Day 10 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.54 B 0.54  B

MAB1

0.57 B 0.80  TO 49

MAB15

0.59 B 0.78  TO Four. Five

MAB17

0.51 B 0.74  TO 37

Table 157: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Tables 158-166 represent TCR analysis of the Rosette Area [cm2] of plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B), are significantly different from those of the control.

Table 158 Table 159

Gen ID

TCR of the Rosette Area [cm2] Normal conditions, Day 3 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

1.73 B 1.73  B

MAB20

2.18 B 3.62  TO 109

MAB43

2.04 B 3.80  TO 119

MAB50

2.25 B 3.81  TO 120

Table 158: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Gen ID

TCR of the Rosette Area [cm2] Normal conditions, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.48 B 0.48  B

MAB2

0.58 TO* 0.70 TO Four. Five

MAB43

0.62 TO 0.75  TO 56

MAB6

0.52 B 0.72  TO fifty

Table 159: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 160

Gen ID

TCR of the Rosette Area [cm2] Normal conditions, Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.84 B 0.84  B

MAB50

0.87 B 0.99  TO 18

MAB6

0.87 B 1.06  TO 26

Table 160: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 161

Gen ID

TCR of the Rosette Area [cm2] Normal conditions, Day 10 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.39 B 0.39  B

MAB10

0.44 B 0.54  TO 37

MAB36

0.45 B 0.51  TO 30

MAB50

 0.45 TO* 0.53 TO 35

MAB6

0.44 B 0.60  TO 51

MAB7

0.43 B 0.50  TO 27

Table 161: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 162

Gen ID

TCR of the Rosette Area [cm2] Normal conditions, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.39 B 0.39  B

MAB20

0.38 B 0.50  TO 28

MAB25

0.39 B 0.53  TO 38

Table 162: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 163 Table 164

Gen ID

TCR of the Rosette Area [cm2] Normal conditions, Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.55 B 0.55  B

MAB10

0.64 TO* 0.71  TO* 30

MAB2

0.63 TO* 0.70 TO 28

MAB20

0.63 TO* 0.67  TO* twenty-one

MAB25

0.64 TO 0.73  TO 32

MAB44

0.65 TO 0.77  TO 41

MAB50

0.70 TO 0.83  TO 51

MAB6

0.63 TO* 0.81 TO 48

Gen ID

TCR of the Rosette Area [cm2] Normal conditions, Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

MAB7

0.61 B 0.73  TO 3. 4

MAB9

0.60 B 0.69  TO 26

Table 163: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Gen ID

TCR of the Rosette Area [cm2] Normal conditions, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.45 B 0.45 B

MAB13

 0.63 TO 0.68 TO* 49

MAB32

 0.50 B 0.74 TO 64

MAB46

 0.52 B 0.75 TO 65

MAB146

 0.64 TO 0.88 TO 94

MAB99

 0.52 B 0.73 TO 61

Table 164: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 165

Gen ID

TCR of the Rosette Area [cm2] Normal conditions, Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.34 B 0.34  B

MAB1

0.36 B 0.45  TO 31

MAB99

0.33 B 0.43  TO 28

Table 165: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 166

Gen ID

TCR of the Rosette Area [cm2] Normal conditions, Day 10 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.66 B 0.66  B

MAB13

0.73 B 0.81  TO 2. 3

MAB3 GA

0.70 B 0.85  TO 29

MAB32

0.70 B 0.86  TO 31

MAB99

0.68 B 0.82  TO 25

Table 166: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Tables 167-175 represent analysis of the Rosette Diameter TCR in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B), are significantly different from those of the control.

Table 167 Table 168

Gen ID

TCR of Rosette Diameter [cm] Normal conditions, Day 3 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.43 B 0.43  B

MAB50

 0.70 TO* 1.50 TO 251

MAB6

0.45 B 1.21  TO 183

Table 167: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means

Gen ID

TCR of Rosette Diameter [cm] Normal conditions, Day 3 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Gen ID

TCR of Rosette Diameter [cm] Normal conditions, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.16 B 0.16  B

MAB10

0.19 TO* 0.21  TO* 28

MAB19

0.20 TO 0.23  TO Four. Five

MAB36

0.18 B 0.21  TO 32

MAB50

0.17 B 0.23  TO 42

MAB6

0.18 B 0.25  TO 57

MAB7

0.18 B 0.24  TO 52

Table 168: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 169

Gen ID

TCR of Rosette Diameter [cm] Normal conditions, Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.25 B 0.25  B

MAB10

0.28 TO 0.30  TO 19

MAB14

0.27 B 0.31  TO 2. 3

MAB19

0.28 TO 0.32  TO 29

MAB2

0.27 B 0.30  TO twenty-one

MAB20

0.27 B 0.29  TO 18

MAB36

 0.27 TO* 0.32 TO 28

MAB43

0.25 B 0.26  B 5

MAB44

0.26 B 0.30  TO twenty-one

MAB50

0.27 B 0.30  TO twenty-one

MAB7

0.28 TO* 0.29 TO 17

MAB9

0.27 TO* 0.30 TO twenty

Table 169: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 170

Gen ID

TCR of Rosette Diameter [cm] Normal conditions, Day 10 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.17 B 0.17  B

MAB19

 0.19 TO* 0.23 TO 31

MAB2

0.20 TO 0.23  TO 32

MAB20

0.19 TO 0.23  TO 33

MAB43

0.19 B 0.21  TO 24

MAB44

0.18 B 0.22  TO 25

MAB50

0.20 TO 0.23  TO 32

MAB6

0.19 TO* 0.24 TO 42

MAB9

0.18 B 0.21  TO 25

Table 170: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 171

Gen ID

TCR of Rosette Diameter [cm] Normal conditions, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.16 B 0.16  B

MAB50

0.19 B 0.22  TO 42

MAB6

0.15 B 0.24  TO 49

Table 171: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 172

Gen ID

TCR of Rosette Diameter [cm] Normal conditions, Day 10 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.22 B 0.22  B

MAB2

0.26 TO* 0.28 TO 27

MAB20

0.26 B 0.30  TO 33

MAB25

0.26 TO* 0.29  TO* 31

MAB43

0.24 B 0.29  TO 29

MAB44

0.25 B 0.29  TO 31

Table 172: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 173

Gen ID

TCR of Rosette Diameter [cm] Normal conditions, Day 3 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.29 B 0.29  B

MAB100

 0.37 TO* 0.51 to 74

MAB13

0.38 TO 0.58  TO 95

MAB15

0.36 TO 0.45  TO 54

MAB18

0.36 TO* 0.38  TO* 28

MAB3_GA

0.43 TO 0.60  TO 105

MAB35

0.39 TO 0.44  TO fifty

MAB46

0.31 B 0.49  TO 65

MAB146

0.35 TO 0.44  TO fifty

Table 173: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 174 Table 175

Gen ID

TCR of Rosette Diameter [cm] Normal conditions, Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.11 B 0.11 B

MAB1

0.13 TO* 0.16 TO 49

MAB13

 0.13 TO* 0.16 TO 41

MAB18

 0.14 TO 0.16 TO Four. Five

MAB32

 0.13 B 0.15 TO 39

MAB146

 0.16 TO 0.19 TO 72

MAB99

 0.12 B 0.15 TO 40

Table 174: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Gen ID

TCR of Rosette Diameter [cm] Normal conditions, Day 10 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.20 B 0.20 B

MAB1

0.25 TO 0.27 TO 30

MAB17

 0.24 TO 0.26 TO* 25

MAB18

 0.25 TO 0.31 TO 51

MAB35

 0.25 TO 0.28 TO 36

MAB146

 0.25 TO 0.28 TO 36

MAB99

 0.24 TO 0.29 TO 44

Table 175: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Tables 176-178 represent TCR analysis of the Foliar Average Area [cm2] in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B), are significantly different from those of the control.

Table 176

Gen ID

TCR of the Foliar Average Area [cm2] Normal conditions, Day 10 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.34 B 0.34  B

MAB10

0.35 B 0.52  TO 56

MAB36

0.40 B 0.52  TO 55

MAB7

0.37 B 0.50  TO 49

Table 176: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 177

Gen ID

TCR of the Foliar Average Area [cm2] Normal conditions, Day 10 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.23 B 0.23 B

MAB13

 0.34 TO* 0.39 TO* 70

MAB146

 0.35 TO* 0.50 TO 117

MAB99

 0.26 B 0.44 TO 89

Table 177: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 178

Gen ID

TCR of the Foliar Average Area [cm2] Normal conditions, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.38 B 0.38  B

MAB10

 0.47 TO 0.51 TO* 35

MAB2

0.41 B 0.49  TO 29

MAB25

0.43 B 0.55  TO 44

MAB50

0.47 TO 0.53  TO 41

MAB7

0.45 TO* 0.50  TO* 31

MAB9

0.43 B 0.54  TO 41

Table 178: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Tables 179-180 represent analysis of the Foliar Average Area TCR [cm2] in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B), are significantly different from those of the control.

Table 179

Gen ID

TCR of the Foliar Average Area [cm2] Normal conditions, Day 5 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.31 B 0.31  B

MAB19

0.35 B 0.48  TO 56

MAB43

0.39 B 0.52  TO 70

MAB6

0.28 B 0.50  TO 62

Table 179: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 180

Gen ID

TCR of the Foliar Average Area [cm2] Normal conditions, Day 8 from planting

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.69 B 0.69  B

MAB14

0.72 B 0.92  TO 32

MAB6

0.69 B 0.96  TO 38

Table 180: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

10 Tables 181-182 represent Dry Weight (PS) analysis by plots in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B), are significantly different from those of the control.

15 Table 181

Gen ID

Dry weight [g] Normal conditions

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

7.75 B 7.75 B

MAB36

 10.37 TO* 13.21 TO 71

Table 181: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 182

Gen ID

Dry weight [g] Normal conditions

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

5.23 B 5.23  B

MAB1

6.81 TO 8.09  TO 55

MAB13

6.08 B 7.61  TO Four. Five

MAB18

6.10 B 8.18  TO 56

MAB99

 6.51 TO* 8.42 TO 61

Table 182: LSM = Mean of least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Tables 183-185 represent analysis of the Weight of 1000 Seeds in plants that overexpress the

20 polynucleotides of the invention under the regulation of promoter 6669. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B), are significantly different from those of the control.

Table 183

Gen ID

Weight of 1000 Seeds [g] Normal conditions

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.02 B 0.02  B

MAB19

0.02 B 0.03  TO 2. 3

MAB2

0.02 B 0.03  TO 44

MAB20

0.02 TO 0.04  TO 71

MAB36

0.02 B 0.03  TO 24

MAB50

0.02 B 0.03  TO 32

MAB6

0.02 B 0.03  TO 22

MAB9

0.02 TO 0.02  TO 19

Table 183: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 184

Gen ID

Weight of 1000 Seeds [g] Normal conditions

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.02 B 0.02  B

MAB20

 0.02 TO* 0.02 TO 17

MAB6

0.02 TO* 0.02 TO twenty-one

Table 184: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 185

Gen ID

Weight of 1000 Seeds [g] Normal conditions

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.02 B 0.02 B

MAB100

 0.02 B 0.02 TO 2. 3

MAB17

 0.02 TO 0.03 TO 33

MAB18

 0.02 B 0.02 TO 18

MAB35

 0.02 B 0.02 TO 28

MAB46

 0.02 TO 0.02 TO twenty-one

MAB99

 0.02 TO 0.03 TO 37

Table 185: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Tables 186-187 represent analysis of Seed Yield per Plant in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669. Each Table represents an independent experiment, using 4 independent events per gene. Genes not related to the same letter as that of

10 control (A, B), are significantly different from those of the control.

Table 186 Table 187

Gen Id

Seed Yield per Plant [g] Normal conditions

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.38 B 0.38 B

MAB1

0.50 B 0.61 TO 61

MAB10

 0.46 B 0.59 TO 53

MAB14

 0.50 TO* 0.60 TO 57

MAB36

 0.52 TO 0.68 TO 77

MAB50

 0.46 B 0.60 TO 56

Table 186: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Gen Id

Seed Yield per Plant [g] Normal conditions

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.32 B 0.32 B

MAB1

0.41 TO* 0.49 TO 53

MAB13

 0.43 TO 0.55 TO 69

MAB18

 0.39 B 0.49 TO 53

MAB32

 0.41 B 0.50 TO 56

MAB35

 0.41 TO* 0.50 TO 57

MAB99

 0.41 TO* 0.51 TO 57

Table 187: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

Table 188 represents analysis of the Harvest Index in plants that overexpress the polynucleotides of the invention under the regulation of promoter 6669. Each Table represents an independent experiment, using 4 independent events per gene. Genes unrelated to the same letter as that of the control (A, B), are significantly different from those of the control.

Table 188

Gen Id

Harvest Index Normal conditions

LSM

Significance LSM of the best event Significance % improvement of the best event

GUI

0.48 B 0.48  B

MAB17

0.46 B 0.62  TO 28

Table 188: LSM = Mean least squares; % improvement = compared to the control (GUI); A means significant difference at P <0.05, A * means significant difference at P <0.1. SEQ ID Nos of the cloned genes (according to the gene ID) that are expressed exogenously in plants are provided in Table 3 above.

EXAMPLE 8

TRANSFORMATION OF M82 TOMATO PLANTS WITH TEAB GENES ASSUMPTIONS

15 For tomato transformation, M82 tomato seeds were previously sterilized with 3% sodium hypochloride + 2-3 drops of Tween 20 (Polysorbate 20). The seeds were washed 3 times with sterile distilled water. The seeds then germinated in 100% Nitsch medium and germinated for 8 days in a culture room at 25 ° C in the dark. Then, the seedlings were cut with a 2-4 cm stem and inserted into 10 cm Petri dishes that were loaded with 30-40 ml of MS liquid medium. Then, the cotyledons were cut and used as

20 explants and subsequently transferred to KCMS solidified medium with 100 µM acetosyringone in a 10 cm Petri dish. The explants were inoculated with A. tumefaciens for 30-50 minutes. The explants were cocultured for 24 hours and transferred to regeneration medium that included Kanamycin as a selection medium. The regenerated resistant seedlings were then transferred to a rooting medium for 1014 days until the appearance of the roots.

25 EXAMPLE 9

GROWTH OF TRANSFORMED M82 TOMATO PLANTS AND PHENOTYPICAL CHARACTERIZATIONS

Experimental Procedures

30 Production of transgenic tomato plants - The plants were transformed as described in Example 8, above. After transformation, the T1 M82 tomato plants were grown until the fruit was established. T2 seeds have been introduced in experiments to assess resistance to abiotic stress.

35 Experimental Results

Trial 1 - Field trial in tomato with water deficit and regular regimes - A field trial in tomato was projected as a drip irrigation system from a source (RGUF) similar to that of a conventional agricultural field. Since the water deficit was applied in a relatively uniform manner, this allowed measuring the effect of drought in populations of small plants. The RGUF method was developed based on an in-line source sprinkler irrigation system (Hanks et al. 1976 Soil Sci. Soc Am. J. 40 pp. 426-429) with some significant modifications. Instead of sprinkler irrigation, drip irrigation was used. To create a layer

deep and uniform wet (at least 60 cm deep), and not the onion-shaped layer that is typically created by drip irrigation, a drip irrigation system with low pressure compensation was used. This system allows to provide small amounts of water in a relatively long time interval. The drought stress field test was conducted on a lighted ground, in an open field (mesh house) near Rehovot, Israel. Between 4 and 5 events were evaluated for each gene and null segregation populations were used as negative controls. During the first three weeks, all plants were grown in a nursery under normal irrigation conditions. After this period, the plants were transplanted according to a commercial cultivation protocol, which maintained a distance of 30 cm between the plants that reached a total density of

2,600 plants per 1000 m2 (the recommended density in commercial crops). Each plant was transplanted near a water irrigation dropper and subsequently underwent two different treatments:

Optimal (100%): optimal irrigation conditions (100%). Irrigation was applied every 2 days as a recommended standard water supply. The recommended standard water supply is the amount applied by local commercial farmers according to standard protocols. Severe stress (50%): Once a day 50% of the amount of optimal water irrigation was applied (at the same time as regular watering was applied). All fertilizers were applied according to local standard protocols. Nitrogen was applied in the same way, as recommended, to all treatments through the irrigation system. Each row, 193 cm wide, contained two drip irrigation lines creating a coverage of six drippers per 1 m2. Irrigation control was performed individually in each treatment. The experiment was structured in a design of 4 randomly distributed blocks, eight plants per plot. The different water regimes began only four weeks after the transplantation of the trees, when the plants began the flowering phase. Water availability in the soil was recorded using tensiometers (used to determine the matrix water potential m that allows assessing the severity of stress). Test 2 - Saline bath experiment in tomato - Transgenic tomato seeds were sown in trays containing denitrified culture medium. Seedlings germinated in nursery conditions. The experimental model of 3 randomized blocks was used, in which 10 plants were planted per event in each block. In the true first sheet phase, the trays were transferred to different "tanks" containing 300 mM NaCl culture solution. For normal treatment, a complete Hoagland solution was applied. Five events were evaluated for each gene, using, as negative controls, nullified segregating populations. The experiment was conducted over a period of 8 weeks, measuring parameters such as chlorophyll content (measured as SPAD units, Soil Plant Analysis Development) and plant biomass (PF and PS).

Although the invention has been described along with its specific embodiments, it is obvious that many alternatives, modifications and variations will appear for those skilled in the art. Therefore, it is intended to include all these alternatives, modifications and variations that are within the spirit and wide scope of the appended claims.

REFERENCES CITED

(Additional references have been cited earlier in this document)

one.

 WWW World Computer Network (.) Fao (.) Org / ag / agl / agll / spush / degrad (.) Htm.

2.

 WWW World Computer Network (.) Fao (.) Org / ag / agl / aglw / watermanagement / introduc (.) Stm.

3.

 McCue KF, Hanson AD (1990). Drought and salt tolerance: towards understanding and application.Trends Biotechnol 8: 358-362.

Four.

 Flowers TJ, Yeo Ar (1995). Breeding for salinity resistance in crop plants: where next? Aust J Plant Physiol 22: 875-884.

5.

 Nguyen BD, Brar DS, Bui BC, Nguyen TV, Pham LN, Nguyen HT (2003). Identification and mapping of the QTL for aluminum tolerance introgressed from the new source, ORYZA RUFIPOGON Griff., Into indica rice (Oryza sativa L.). Theor Appl Genet. 106: 583-93.

6.

 Sanchez AC, Subudhi PK, Rosenow DT, Nguyen HT (2002). Mapping QTLs associated with drought resistance in sorghum (Sorghum bicolor L. Moench). Plant Mol Biol. 48: 713-26.

7.

 Quesada V, García-Martínez S, Piqueras P, Ponce MR, Micol JL (2002). Genetic architecture of NaCl tolerance in Arabidopsis. Plant Physiol 130: 951-963.

8.

 Apse MP, Blumwald E (2002). Engineering salt tolerance in plants. Curr Opin Biotechnol. 13: 146-150.

9.

 Rontein D, Basset G, Hanson AD (2002). Metabolic engineering of osmoprotectant accumulation in plants.Metab Eng 4: 49-56

10.

 Clough SJ, Bent AF (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735-43.

eleven.

 Desfeux C, Clough SJ, Bent AF (2000). Female reproductive tissues are the primary target of Agrobacterium mediated transformation by the Arabidopsis floral-dip method. Plant Physiol 123: 895-904.

CD-ROM Content (Compact Disc For Reading Only)

Below is the list of the contents of the files on the CD-ROM that is attached with this report and is presented with the application. File information is provided as: file name / size in bytes / creation date / operating system / computer format.

CD-ROM1 (1 SEQUENCE LIST file):

1. “43562 Sequence Listing.txt” / 3,856,000 bytes / July 24, 2008 / Microsoft Windows XP Professional / PC.

Claims (9)

one.

 A method to increase biomass, growth rate, seed yield and / or tolerance to abiotic stress of a plant compared to biomass, growth rate, seed yield and / or tolerance to abiotic stress of a non-transgenic plant, in which abiotic stress is selected from the group consisting of osmotic stress, saline stress and nitrogen deficit, the method comprising overexpression in the plant of an exogenous polynucleotide encoding the polypeptide SEQ ID NO. : 201 or an ortholog thereof comprising an amino acid sequence having a homology of at least 80% with the amino acid sequence set forth in SEQ ID NO: 201, wherein said polypeptide is capable of increasing biomass, the rate of growth, seed yield and / or tolerance to abiotic stress of a plant, in which abiotic stress is selected from the group consisting of osmotic stress or, saline stress and nitrogen deficit, wherein said exogenous polynucleotide is comprised in a nucleotide acid construct that additionally comprises a promoter to direct transcription of said exogenous polynucleotide in a cell of a plant, said promoter being a constitutive promoter, a specific tissue promoter, or a promoter inducible by abiotic stress, thereby increasing biomass, growth rate, seed yield and / or tolerance to the abiotic stress of the plant compared to biomass, the rate of growth, seed yield and / or abiotic stress tolerance of the non-transgenic plant.

2.

 The method of claim 1, wherein said amino acid sequence has a homology of at least 86% with the amino acid sequence set forth in SEQ ID NO: 201.

3.

 The method of claim 1, wherein said amino acid sequence has a homology of at least 95% with the amino acid sequence set forth in SEQ ID NO: 201.

Four.

 The method of any of the preceding claims, wherein said exogenous polynucleotide encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 201.

5.

 The method of any of the preceding claims, wherein said promoter is a constitutive promoter.

6.

 The method of any of the preceding claims, wherein said exogenous polynucleotide is set forth in SEQ ID NO: 1530, 1 or 1531.

7.

 The method of any of the preceding claims, wherein said polynucleotide is heterologous to said plant.

8.

 The method of any of the preceding claims, further comprising cultivating the plant expressing said exogenous polynucleotide under abiotic stress.

9.

 The method of claim 1, wherein said ortholog comprises the amino acid sequence set forth in SEQ ID NO: 289.